BIOLOGY INTERNATIONAL€¦ · Biology International No 35 (August, 1997) Tumors in Lungfish The...

BIOLOGY INTERNATIONAL The News Magazine of the International Union of Biological Sciences (IUBS)

CONTENTS (NG 35,1997) .- - ;TT!:- , - . .I 1. -

EDlTORl AL The Prince Hitachi Prize for Comparative Oncology

FEATURE ARTICLE Biodiversity in Fish Tumors By Prince Masahito and Takatoshi Ishikawa, Japan Crop Residues as a Resource: The use of fungi to upgrade lignocellulosic wastes By Joan Kelley and Russell Paterson

SPECIAL FEATURE: BIOSYSTEMATICS Biosystematics: Meeting the Demand By D. L. Hawksworth The Perspective of the Systematist By B. R. Baum The Perspective of the Biodiversity Prospector By H. G. Wildman The BioNET-INTERNATIONAL Approach By T. Jones The European Network on Systematics Biology By W. Los and S. Blackmore

NEWS HlGHLlGHTS Obituary: Gaetano Salvatore (1 932-1 997) IUBS 1996 Financial Statement

PUBLICATIONS REVIEW

CALENDAR OF MEETINGS

Editorial

The Prince Hitachi Prize for Comparative Oncology

Tumors or cancers occur as an outcome of changes intrinsically associated with cellular properties of ri11 forms of organisms. The comparative views of them should not be overlooked as a problem of biology per se. Comparative oncology, however is an unjustly neglected area, in spite of its abundance of interesting and important subjects. Stiidies i n this area have its own merit to look for the evolutionary origin of tu~iiors and for coniprehending the distribution of their occurrence i n diverse orgrinisms. In turn, these studies are well expected to open a new breakthrough for oiir linderstanding of a nature of tumorgenesis. They may also provide opportui-iities to establish new mode1 systems for unique approaches to cancer studies of higher aniil-ials including human beings. Therefore, the establishment of the Prince Hitachi Prize for Comparative Oncology in 1995 by the Japanese Foundatioii of Cancer Research is justly timely.

The prize wris iiistituted i n con-in-iemoration of the sixtieth birthday of Prince Hitachi who has long been devoted himself to researches in this field. His Imperia1 Highness Prince Hitrichi, (Prince Masahito) intierits his great interest in biology fron-i his fiither, the late Enîperor Showa who was internationally highly respected for his ei-iiinent contributions to taxononiy studies of some groups of marine invertebrrites. An iiiterest of Prince Hitachi was oriented towards ce11 biology, and since 1969 he hris been studying the problems related to cancer at the Cancer Institute, the Japriiiese Fouiidatiori for Cancer Research, as guest researcher.

In 1995, in con-iniemorrition of Prince Hitachi's sixtieth birthday, Haruo Sugano and Takatoshi Ishikawa, liis supervisors and collaborators, compiled al1 his publications so far printed. This compilation contains 56 papers, 40 of which are written in English. Although some subjects cover various areas of biology, most notable and central are the contributions to tumor studies on lower vertebrates such as fishes and frogs. For example, a discovery of erythrophoromas in gold fish is fanious and a colour photograph of this precious fish with red-coloured skin tumor decorated the cover of the Journal of National Cancer Institute (1978). Studies on nephroblastomas occurring in Jrzprinese eels are of particular interest, because Prince Hitachi not only discovered the tuinors, but succeeded i n showing that their incidence is dependrint on water tenipernture and popiilation density.

Biology International No 35 (August, 1997)

Comparative onçology is certainly one of the important sectors in the study of biodiversity. Readers of the present Journal will have a special attention to his discovery of tumors of lungfish. He found that an incidence of tumor is relatively high in this "living fossil" fish.

It is needless to say that IUBS is honoured and pleased to receive the article by Prince Hitachi i n this officia1 journal. 1 personally hope that many readers will recognize the importance and interest of comparative oncology which seems to be underrated in both biologiçril and medical sciences.

Tokindo S. Okada (Vice President, IüBS) JT Biohistory Research hall

Murasaki-cho, Takatsuki, Japan

Awardees of Prince Hitachi Prize are:

1996 Professor John C.. Harshbarger (The George Washington University, Medical Center, Washington, D.C., USA).

1997 Professor Fritz W. Anders (Genetisches Institut, Justus-Liebig- Universitat, Giessen, Germany).

For more information concerning the Prize, piease write to:

The Executive office The Prince Hitachi Prize for Comparative Oncology Japanese Foundation for Cancer research, 1-37-1 Kami-lkebukuro, Toshima-ku, Tokyo 170 Japan. Tel: 03-39 18-01 1 1 ; Fax: 03-391 7-7564.


Biodiversity in Fish Tumors By Prince ~asahi to* and Takatoshi lshikawa"

*. Department of Pathology, Cancer Institute, Kami-lkebukuro, Toshima-ku, Tokyo 170 " Faculty of Medicine University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113

Prologue

Prince Masahito, the first aiithor of this review, began his career in Cancer Research in 1968 under the direction of Dr. Tonîizo Yoshida, a scholar of world wide renown who was then the director of the Cancer Institute. His stress on the individuality (biodiversity) of tumor cells is based on the detailed observation of the Yoshida sarcoma. In 1982 Prince Masahito commenced cooperative studies on fish tumors with Dr. Ishikawa, who had obtained his doctorate for studies on the induction of hepatocellular crircinomüs in medaka, a species of bony fish, using the carcinogen diethyliiitrosari~ine, DENA (Ishikawa et al., 1975).

Generally speaking fishes are divided into four classes, that is, hagfishes, lampreys, canilaginous fishes and bony fishes (Robins et al., 1991) Among these bony fishes are the most species rich of a11 the vertebrate group, with over 23,000 species described (Nelson, 1994). This displays great diversity in behaviour, habitat and morphology occurring i n al1 water bodies on the surface of the globe. They are even found in hot soda lakes that have a temperature as high as 44 Co and under the Antarctic ice sheet at about -22C0 (Nelson, 1994)

This bony fish diversity means that they provide an ideal mode1 for investigation of cancer diversity. For exanîple, studies of tumors in lower aquatic animals have now gained considernble importance as a method for detecting injurious agents in the environment. Investigation of fish tumors is also a basic biological approach to understünding possible nîechanisn~s of carcinogenesis (Prince Masahito et al., 1986).

Having niade these comnîents regarding the rational for investigating fish tumors for biodiversity of cancer, the authors would like to turn to specific examples, and particulnrly to work in which we have participated.

Historical Note on Fish Tumor Studies

The first reported fish tumors were osteomas in several pinnate batfish, Platax pinnatus in 1793. As osteomas in fish are often inflammatory, a fibroma , noted in a common cürp reported i n 1854 might be the oldest documented example of a


bony fish tumor (Wellings, 1969). The earliest fish tumors studies were reviewed by Schlumberger and Luché (1948), Wellings (1969) and Mawdesley-Thomas (1975) in detail. Attention was concentrated on two former reviews on histological types of lesions while the latter described findings in fish family. The commonest neoplasmsin bony fish were initially found to be in skin papillomas or carcinomas (Schlumberger and Luché, 1948; Wellings 1969). Subsequently in the Registry of Tumors in Lower Aniil-irils (RTLA) report pointed to peripheral nervous systems tumours as the cornmoilest fish neoplasms (Harshbarger et al., 1981). Fibromas or fibrosarcomas also frecluently observed in bony fish (Wellings, 1969). On the other hand, turnors of the stomlich, intestine and swim bladder, the homologus organ to the mammrilian lurig are rare (Wellings, 1969). In general in bony fishes which belong to the Cyprinidrie and Salmonidae fish tumors are commonest (Madesley- Thomas, 1975)

No review of fish neopliisia would be complete without mentioning the pioneering efforts of the Keizo Takahashi, Louis Thomas and Clyde J. Dawe, who devoted their concesils to the study of fish tuinors. Dr. Takahashi, a pathologist who was employed by Nigiita Medical College, reported 155 cases of neoplastic lesions arnong 100,000 fish caiight rilong the Japanese coast between 1925 and 1950 (see Takahashi, 1929 for :in e;trly description.) Al1 fishes , anlphibians and reptiles have three kinds of pigment cells, namely melanophores, bright coloured pigment cells (xanthophores and erythrophores) and iridescence-producing cells (iridophores or guanophores) and tiimors with three distinct phenotypes might therefore be expected to develop f~-0111 the corresponding pigment ce11 types, (Ishikawa et al., 1978) true enough Dr. Takahashi found melanon~as in 2 walleye pollock, Theragra clzalcograrnrnu, and a club rnackerel, Scomber juponicus, an erythrophoroma in a hobo gurnard, CI-~elidorzicl~tlrys ,spino.su,s, and a guanophororna in 1 greeling, Hexagrum~non.~ otukii (Takahashi, 1929; Wellings, 1969). Dr. Louis Thomas strirted his own collection of fish tiin~ors at the two islands of St. Pierre and Micluelon, French territories near Canada i n 1923 (Thomas, 1931). He first found an olhctory neuroepithelioma i i i one "daurade" (French common name) in 1932 (Thomas, 1932). The "driurade" was recorded as a gilthead, Sparus auratus in the review by Schlumberger rind Luché (1948). But this nlay not have been correct, since this fish is not generrilly found around Canada. Dr. Clyde J. Dawe who was with the Laborütory of P;ithology, Niitionril Cancer Institute, USA, left a particularly indelible work in the field of coinprirative oricology, particularly with his establishment of RTLA with the help of Harshbarger, at the Smithsonoian Institution i n 1965. Specin~ens continue to be collected from at least 25 countries for analysis rit the preseiit d;iy. One result is that we now know that tumors are far more coilimon in bony fishes (at an incidence of about SI%), than in sub- hon~eothennic Chordata where they are comparatively rare (0-1 1 %) (Harshbarger et al., 1981 ). th;inks to the RTLA and the efforts of Dawe and Harshbarger, the study of fish tiirnors has greatly advanced. A case of an olfactory neuroepithelioma found in a bloater, Coregonus koyi was one of the exciting highlights of the history of the RTLA (Dawe rind Hlirshbarger, 1975).


Tumors in Lungfish

The question of whether tumors n-iight develop in species which show a very slow rate of evolutionary change was raised in connection in the role of DNA mutations in neoplasia (Dawe and Harshbarger, 1975). In this context, lungfishes which are considered to be survivors from the Lower Devonian with almost no morphological changes occurring since then (Nelson, 1994), are clearly of interest. The present authors therefore started to look for tumor bearing fishes in cooperation with aquariums between 1984 and 1986 and found 5 out of 14 dead or morbund fishes to have lesions (Prince Masahito et al., 1984, 1986b; Ishikawa et al., 1986). These included two speriî~atocytic seminomas (Prince Masahito et al., 1984), 5 liver tumors (Prince Mus;ihito et al., 1986) and 2 neurinomas (Ishikawa et al., 1986), as listed i n Table 1. Multiple dark green nodules in the liver after formalin fixation were histopatho1ogic;illy established to be hepatocelluler carcinomas in Protopter~ls umpl~ibiu.~ (Prince Müsahito et al., 1986). Nigrelli and Jakowska (1953) ülso reported a spermritocytoma and a renal melanoma in Protopterus annectens. An intestinal melanosarcorna was recorded in one albino lungfish, Protopter~ls dolloi (Harshbarger, 1979 , and a seminoma and a leiomyosarcoma were found in rinother (Hiibbard and Fletcher, 1985) Falkmer et al., (1977) also noted a high incidence of liver tun~ors in the Atlantic hagfish, Myxine glutinosa, a merriber of a second prii-i-iitive class of fish with a fossil record stretching back about 300 n~illion ye;irs (Nelson, 1994). Thus it is clear that the potential for tumor developrnent is iiot eliriiirinted by an extremely slow rate of evolutionary change (Prince Mas~ihito et al., 1986)

Table 1. Tumors in Lungfish. (* P.= Protopterus, L.= Lepidosiren)

5

species* No. of Period in Tumors Reference Fish Aquarium

(yr.1 P. annecretzs

P. dolloi

P uethiopiciis

P. dolloi

L. paradoxu

L. paradoxa

P . amphihius

P. annecteus +

Spcrina~ocy~oina & rcnal mclanoina intcslinal mclanosarcoina spcrinalocy lie scminoina scin inoma lciom yosarcoma Hcpa~occllular carcinoma Hepa~occllular carcinoma Hcpalocellular carcinoina Ncurinoma

Nigr~lli & Jakowska 1953 Harshbarger, 1976 Prince Masahito et al., 1984 Hubbard & Flctchcr, 1985 Prince Masahito et al., 1986 Prince Masahito et al., 1986 Prince Masahito et al., 1986 Ishikawa et al., 1986

1

1

1

1

1

1

1

1

5

7

9

y

5

2

4

4


Chemical Carcinogenesis in Fish

Hepatocellular carcinom:is were first recorded in two rainbow trouts of the genus, Oncorhyncus mykiss by Hriddow and Blake (1933). Cottonseed meal was introduced as feed for rainbow trout, used in California in 1936, instead of fresh meat and fish (Wales, 1970), and thereafter the number of reported liver tumors gradually increased, becorning widespread in various parts of the world by the late 1950's (Wales 1970). Wolf and Jackson (1963) established a direct relation with cottonseed nieal feeding and Sinnhuber et al. (1968) demonstrated induction of hepatocellulrir carcinotîîas in trout fed aflatoxin-contaminated samples. Wales (1970) in fact showed that the rainbow trout is the most sensitive to aflatoxins of al1 animals including rodents, with exposure to only 20 ppb of aflatoxin Bi for one day resiilting in tiirnos development. He also denionstrated that fish differ greatly in their susceptibility to aflntoxiti B I , even anlong Salmonid species (Table 2)

Table 2. Susceptibility of Salmonid Fishes to Aflatoxin B I (Data compiled by Wales, J.H., 1970)

Species Dose level (ppb) Observation Hepatoma Period (mos.) Incidence

Stanton (1965) wis the first to succeed i n inducing liver tuniors, in Branchodanio rerio. Japanese reserirchers, including the present authors subsequently reported that medaka, 0ryzia.s lutipes is very sensitive to DENA (Ishikawa et al., 1975; Ishikawa and Takayania, 1979). In contrasts they failed to induce liver tumors with this carcinogen i n slendes bitterlings, Acheilognathus Iunceolatus, treated in a similar manner (Trikayama et ul., 1981). Aoki and Matsudaira (1977, 1981) reported the induction of liver tunîors in niedakas using methyl azoxy methanol (MAM) acetate. Surprisingly, both hepatocellular carcinonias or cholangiomas were found in 50 to 60% of medaka 60 to 90 days after treatment with 2.0 to 3.0 ppnî MAM acetate for only one day.

100%

90% 14% 0% 0%

medium

medium 28% low 0% 50%

Rainbow trout Mt. Shasta Donrildson's Kaniloops Steelherid Golden trout Cutthroat trout Coastal Lahontan Brook troout Chinook saliîion Coho salnion Soçkeye s;ilnion

12

12 8 8 8 '?

O

7 O

7 12

8

12 12 9 9 ')

? 1 O ?

1 O 12


Virus-Associated Tumors of Fish

A role for both DNA and RNA viruses as etiologic agents for bony fish neoplasms has been established i n a few cases. Enzootic lymphomas found in the northern pike, Esox 1~~ciu.s i n the United States of America (Dawe and Harshbarger, 1975), Ireland (Mulcahy, 1976) and Sweden (Ljungberg, 1976) were transplantable with evidence of ce11 -free transmission (Mulcahy, 1976; Mulcahy and O'Leary, 1970). The presence of reverse transcriptase and C-type virus like particles was also docuniented for these lyniphonias (Papas et al., 1976, 1977) In the muskellunge, Exos mu.syuitzorzgy, of the United States of America and Canada epizootic lymphomas were sii-i-iil;irly reported. (Dawe and Harshbarger, 1975; Sonstegard, 1975)

Investigation of the plasmncytoid leukemias which caused severe losses in chinook salnion, Otzcorl~y~zcllu,~ t.sl~uwyt.scha reared i n British Columbia, Canada, demonstrated the disease could be transmitted experimentally to healthy fish by intraperitone~il injection of kidney tissue from a tumor bearing animal. Al1 25 sockeye salnion, O~zcorhy~zch~is nerka, treated i n this way developed leukemias within 10 weeks rifter exposure, while 26 rainbow trouts were not affected (Kent and Dawe, S.C., 1990) Lüter, ce11 free transmission was also demonstrated (Eaton et al. 1993) l n chinook stilmoii with the leuken-iia, reverse transcriptase activity and C-type virus like particles were found (Earori and Kent 1992). The first isolation of C-type virus from ri s;ili-i-io~iid fish rapidly followed (Eaton et al., 1993)

With regard to DNA viruses, Kiniiirri. et al., (1981) found a herpes virus in the ovarian fluid of normal yrimame, land-locked masu salmon, Oncorynchus masu. and nanied i t accordingly as the Otzcorlzynclzus masu virus (OMV). When the OMV was tmnsniitted artificially to 3-5 nionth chum salmon, Oncorhynchus keta , they began to die 11 to 12 days later and 35 to 60 % of them succumbed within 60 days due to liver necrosis. Skin tuniors, mostly in the oral area, developed among survivors from 4.3 to 8 moiiths post artificial infection. The viruses could be successfully recovered froiii inost derid salmon. Siniilar tumors were also inducible in the coho salinoii, Otlcorllyncllus kis~itclz, rainbow trout and sockeye salmon. Sano et al., (1983) also isolrited ri herpes virus termed the yamame tumor virus, YTV, from a sporitnneous mandible tunior in yamanie. The chuni salmon was aIso susceptible to YTV. Hedrick et ul. (1987) and Yoshimizu et al. (1995) subsequently proved tlirtt OMV riiid YTV are one and the same.

Another exrimple of u DNA virils, the cyprinid herpes virus, CHY, was isolated frotii 9 papillonias developing on the skin or caudal fins of Japanese fancy carp (Sano et al., 1985). When the comnion crirp, Cyprinus carpio were placed in water containing CHV iit 20C0, they strirted to die within 3 to 4 weeks (Sano et al., 1991). The papilloinris apperired rifter 5 to 6 nionths in survivors, and the CHV could be reisolrited froiii al1 cnses.However, this virus hrid no effect on cyprinid fishes other than the coiiirnon riiid fiiricy carp.

Biology international No 35 (August, 1997)

Pigment Cell Tumors

In the RTLA report (Harshbarger et ul., 198 1) it was noted that pigment ce11 tumors were aniong the coinmoriest neoplnsirs arising in bony fishes, next to peripheral nerve tumors. I n addition to typical melanomas or melanophoromas, there are peculiar neoplasms (erythrophoromas and iridophoromas) in fish which do not have counterparts in maniiiîals.

In fish the term melanophore is conlnionly applied instead of melanocyte, because of the presence of niobile pigment granules in the cytoplasm (Bagnara and Hadley, 1973) Therefore sonie cori~piirative oncologists prefer to use melanophoromas to designate fish nielarion~;is. Siich melrinocyte-delived neoplasms are the commonest pigment ce11 tiiiiiors iri fish (Hnrshbarger et al., 1981). They are primarily located beneath the b:isement 1;iniiria of the skin (Bagnara rind Hadley, 1973)

Genetically determined melanonia developnient in hybrids between the green swordtail, Xipl~opl~o1li.s Ilelleri and the southern platyfish, X. maculatus was first observed by Haussler (1928) and von Kosswig (1928). Reed and Gordon (1931) systernatically aiialysed the uriderlyiiig genetic factors and considered melanomas occurring in the hybrids ro origiiiate from black spots (macromelanophores) inherited from the plliryfish fish followirig Medelian laws (Haussler, 1928; von Kosswig, 1928; Reed and Gordoil , 1 93 1, Gordon, 1947). Macromelanophores are lacking iri the swordtnil. The progression from back spots to melanomas was later proposed to be ii resiilt of enhriiiced color gene expression i n the pigment cells (Anders et al. 1976) . The color gene, nariied tunior gene (Tu) as an oncogene by definitiori (Anders et al., 1976), is normally under negative control by linked and non linked regiilatory geries (R genes). If Tu is lacking, no melanomas occur while if the R genes are still present i n the hybrids, benign, malignant lesions develop. If sonie R genes are still present i n the hybrids, benign nielanomas occur (Anders et al., 1976) The Tu geiie is locrited on sex (X or Y) chromoson~es and the R genes on an autosome (Ahuja et ul., 1980) Wittbsodt et al. (1989) cloned the melanoma inducing gene (Xmrk) frorn the TLL lociis of platyfish and established that it encodes a novel receptor tyrosine kinase which is closely related to the EGF-receptor.

Chemiciil cnsciiiogens niriy ulso play an important role in nielanoma induction in fish. Hyodo-Tiigiiclii riiid Matsudaira (1984) succeeded in iriducing transplantable nielanomas by treatiiierit witli N-niethyl-Nt-nitsosoguanidine in an inbred strain of medakri, 0ryziu.s L~~ti11e.s. Extensive investigating of the geographic distribution of nielanoniris in the iiibe csoaker, Nibeu mitsukuri along the pacific Coast of japan, showed niark varititioii i n turiior incidence ranging from zero to 47 % at each different place (Kimiira, 1. et ul. 1984). The authors therefor concluded that environmentril fiictors are involved i n active induction of the melanomas, these most likely being cliemicnl carcinogens.


Erythrophoron~as or x;intophronlas arise froni erythrophores or xantophores (Ishikawa et ul., 1978) In the goldfish, these are located in the derniis just beneath the basemeiit membrane (Matsumoto and Obika, 1968). Under the electron microscope their cytoplasm appears occupied by a large number of round membrane-bouiided organelles, called pterinosomes. The bright color of individual cells is detemiiried by the kinds and amounts of individual pigments. For example, the Iarval x;iritophore pigment of goldfish is yellow due to sepiapterins and erythrophores i n xiphophorin fishes contain red drosopterins. Carotinoid are responsible for the sed color of adult goldfish (Matsumoto and Obika, 1968)

Smith G. M. (1934) first reported a cutaneous erythrophorama in winter flounder, Pleuronectes uniericunus. I t is difficult to diagnose such lesions purely on. the basis of histologic examination because there are sinlilarities in histologic patterns to cutaneous tumors in fish. In erythrophoromas in goldfish, tumor cells are arranged in whorled structures or parrillel rows with palisading nuclei, which are also characteristic of peripheral nervous systenl tumors. Therefore, based on observations of erythrophron~ii-beriring 17 goldfishes (Ishikawa et al., 1978) the present authors coiisider th;it pigment analysis of the tumor ce11 is necessary for accurate diagiiosis of erythi-ophromas or xantophoromas using electron rnicroscopy or biochen1ic;tl metliods. Etoh et al, (1983) reported that the incidence of erythrophorom;is iiicserised with age, fin~illy attaining a level of about 60% in the oldest of their stock.

Iridophoromas (gurinophoror~ias) are neoplastic growth of iridiscent pigment cells, termed iridophores or guriiiophores, located in the dermis and responsible for the silver or blue iridscence so characteristic of the fish coloration exhibited by the skin of the sprat, Spruttus spruttus, horse mackerel, Truchurus trachurus and neon tetra, Hyphe.s.sohryc~~orz itztlesi. These cells contain crystals of guanine, which constitute the interna1 components of reflecting platelets, pigment organelles (Denton and Land, 197 1 )

Iridopohoromas rire rare :is conip:ised with melanotnas and erythrophromas (Schlumberger ;ind Luché, 1948; Wellings, 1969; Mawdsley-Thomas, 1975; Harshbrirger et ul., 1981). However, since they feature characteristic intra- cytoplasniic reflectiiig platelets, they can be easily distinguished from other subcutaneous tutnors nt both the light aiid electron nlicroscope levels.

The first exriniple of iridophoroma wrts recorded by Takahashi (1929), found on the left side of the caudal fin of a greenling, Hexugrummo.~ otakii and presenting silver white with a peci11i;ii. sheeii. Kiniiira et ul. (1984) reported a nuniber of interesting cases of iridophoroinns i n iiibe croakess which were complicated with melanoma developmen t withiii ttie sanie iiodiiles.

The leiicophore, has receiitly beconle widely recognized as an independent pigment ce11 by pignient ce11 researchers. However since they are only found in a few fish


species such as the medrika (Obika, 1988) "leucophollomas" theoretically expected to originate from leucophores have not yet been reported.

Epilogue: Oncogenes in Fish Tumors

Oncogenes, hrive beconie a major focus of attention in the cancer research. As stated in this review, the tuniors in bony fish are as diverse as those in mammalia. However, the available information concerning oncogenes in fish tumors is still limited. One siirprising fiict which has enierged is that fish oncogenes are highly conserved rit the amirio acid and nucleotide sequence levels among bony fishes otherwise hnvirig niarked species diversity.

Src-gene: Expressioii of the src gerie has been exaniined in various bony fishes using anrilysis of 60-kd phosphoprotein product (pp 6 0 ~ ~ ~ ) - s ~ e c i f i c activity (Barnekow et ul., 1982; Schnrtl rind Bririiekow, 1982; Barnekow and Schartl, 1987). The highest kinase lictivity was detected in extracts from brains and melanoinas of plntyfish-swordtriil hybrids among bony fishes, with specific pp 60°C activity lower i n benign ris compüred with nialignant tumors (Barnekow et al., 1982). Barriekow et al., ( 1 982) confim-ied the presence of pp60c-SrC associated kinase in bniin extracts of 10 kinds of normal killifishes. Some activity was also fourid in normal skiri, livers, spleen and testes, but not muscle tissue (Barnekow et al., 1982). Activity lias si~~iilarly been detected in the plaice, Pleuronectesplatessa, tub gurniird, Trigla l~~cerna, river gludgenon, Gobio gobio, atlantic codfish, Gadus morhuu, flounder, P1acicl~cy.s j'lesu.~, European roach, Leuciscus rutilus, convict cichlid, Cicl~lu.sornu rrigroji~.sciuc~~rn and Europeün eel, Anguila anguila (Schartl and Barnekow, 1982; Barnekow and Schartl, 1987).

Yes and fin-genes: cDNA clones and X-yes and X-fin genes, related to X-src, in the normal swordtail showed 80-90% homology in exons 3 to 6 and exons 7 to 12 at the predicted amino :teid level to their human counterparts. Exons 3 to 6 contain conserved regiilatory donirlins. The yes and fin-genes were also found to show the highest expression i n miilignrint nielrinonias in platyfish-swordtail hybrids (Hanning et ul., 199 1).

Myc-geiie: The rainbow trout c-~iiyc wris cloned and sequenced by van Beneden et al. (1986) rind thus reveriled to have extensive honiology to the c-myc genes of chicken, niouse and man. Two domains i n the second exon in particular demonstrate about 90% honiology at the amino acid level. Fish c-myc genes have also been cloned rind secliienced for the zebrafish, Branchyodanio rerio (Schreiber- Agus et al., 1993), goldfish (Zhring et ul., 1994) and carp, Cyprinus curpio (Zhang et ul., 1993). The zebta fish c-niyc gene proved able to activate H-ras and to effect malignant transforniation of n~animaliaii calls (Schreiber-Agus et al., 1993). Schreibes-Agiis et ul. (1993) observed expression of the zebrafish L-myc to be connected with early enibryogenesis while the zebrafish N-myc exhibited a relation to growth of easly orgcins. They also reported steady-state mRNA expression of c- mye in diffei-entinted tissue.


Ras-geiie: The normal goldfish rus gene wris cloned and sequenced by Nemoto et al. (1986), in the ri~itliors' group. This was the first fish oncogene cloned along with the rüinbow trout c-niyc gene (Van Beneden et al., 1986), and showed 96% homology with the humon K ras gene at the amino acid sequence level. The degrees of homology between the goldfish ras gene and human H-ras and N-ras genes were 87 to 88%. I n goldfish erythrophoronia permanent ce11 lines were established by Matsumoto et ul. (1980). No point mutations were observed in the 12th codon (Neiiioto et al., 1987)

An extre~nely high incidence (55-90%) of hepatocellular carcinomas in adult tonicod, Microgudu.~ tomcod, in the Hudson River has been documented (Wirgin al., 1989) Wirgin et (11. ( 1 989) presented evidence of an exogenous K-ras gene in the toincod liver tiiniors by soiithern blot analysis of al1 primary (616) transfectants using NIH3T3 aiid niide iiioiise tumor DNAs. But, no activation of the H-ras gene was detected. They fiiiled to obtain any hybridizrttion signals when tonîcod liver DNA was hybridized with the cloned goldfish rus gene.

When DNA froiii 7 oiit of 13 liver lesions of winter flounders including hepatocellular and cliol:irigiocellulrir c;ircinonîas, was transfected into NIH3T3 niouse fibrob1;ists (McM:ihon et ul., 1990) subcutaneous fibrosarcomas appeared in athyniic niide iiiice. The presence of winter flourider K-ras gene was confirmed in al1 10 fibros;ircoiii:is by soiithern blot niialysis. Analysis of the K ras gene denionstr;ited miit:itioiis iiivolvirig GC->AT or GC->TA single base changes i n the 12th codon but no a1ter;itioiis i n the 59th codon.

Two rus geiies, desigiinted trout rus- l and rus -2, have been cloned and sequenced from a noriii;il rainbow trout liver cDNA library (Mangold et al., 1991). The predicted :inlino-;icid secllience of the trout rus-1 gene differs from human K-ras in only one of the 172 rtniino acid sesidues, with a change from serine to threonine at position 136. The aiithors coricluded that trout rus-1 gene is a K-ras gene.

Chang et ul., (1991) an:ilysed rus gerie miit;ttions in rüinbow trout hepatocellular carcinomas and inixed Iiep~itocell~il:ir/cholangiocell~i1ar carciriomas induced by aflatoxin BI iising the polyiiierase chLiin seaction and oligonucleotide hybridiation methods. Tlie results witli the two rnetliods were the san-ie. Point mutations in exon 1 of the first troiit rus gene were detected in IO out of 14 aflatoxin Bi induced liver tuniors. Codon 12 GGA -> GTA trnnsversions was found iri 7 cases and codon 13 CGT-> GTT tr;iiisvessioiis i n 2. In 1 case, a codon 12 GGA-> AGA change was detected. However, no poirit iniit:itioiis were observed in exon 1 of the second trout ras gene. These resiilts are reniiniscent of the findirig of point mutations of K-ras gene in r;it liver tiiiiiors indiiced by af1l;itoxiii B 1.

The ras geiie is widely coiiserved in nî;iny kinds of eukaryotes examined, including manininls, chickens, fisli, iiiollusks, drospophila, plants, slinie molds and yeasts. This fact siiggests that i t in:iy play a fiind;iiîîerital role in cellular physiologies retriiiiing iiiiportunt bio1ogic;il fiinctioris throughout evoliitioii (Barbacid, 1987)


p53 gene: Smith et ul. (1988) have revealed that a sequence homologus t o the man~rnrilian p 53 is present i n the carp (epit~~eliomapapillo.sum cyprini, EPC) and a ch inook salnion e inbryo (CHSE 214) ce11 l ine we re dot-blot a n d southern-blot analyses.

Caron d e Fromental et (11. (1992) cloned and sequenced a p53-encoding c D N A o f rainbow trout rind demoiistrüted ü 47% sinlilarity to t he human and Xenopus laevis p 5 3 genes. The encoded p 5 3 protein features conserved domains , an ac id ic N temlinus and ri hydsophillic C temlinus can specifically interact with the SV40 large T antigen. Clearly m o r e attention t o the role of oncogene changes in generation o f fish tuniors is w~irsanted.

Acknowledgements

Wc would likc to iliank Dr. t l . Sugano, honorary dircctor of the Canccr Institute, Tokyo, Jiipaii For Iiis kiiid ciicouragcincnl and advicc. WC arc gratcful 10 Dr. K. Mlilsuura, cur-alor, tlic N:itioiial Scicncc Muscuin, Tokyo, Japan for his carcful chcck of scicniil'ic Iïsh n;iii-ics. Wc also tliank Miss T. Nanuoshi for sccrclarial hclp in thc prcpa~i t ion l'os in:inusci~ipt. The dcscribcd rcscarch was supportcd by Grdnls-in Aid for Canccr Rcsc;iscli l'rom tlic Minislry of Educalion, Scicncc Sporls and Cullurc, and Lhc Ministry ol' t1c;iltli aiid Wc1l';isc.

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Biology lnternational No 35 (August 1997)

Crop Residues as a Resource The use of fungi to upgrade lignocellulosic wastes

by J o a n Kelley a n d Russell Paterson International Mycological Institute, Bakeham Lane, Egham Surrey TW20 9TY, UK

Introduction

Large amounts of renewable lignocellulosic residues are generated annually both naturally and as a result of agricultural and industrial activities. The world annual production is assessed as 20-50 x lo9 tonnes of which 4 x 109 tonnes are available for use. The 'wastes' show considerable potential for exploitation as they contain cellulose, hemicelluloses and lignin. I-Iowever, the crucial technical limitation is the digestibility of the residues, which is approximately the relative proportion of lignin to cellulose. A high lignin to cellulose ratio implies that the residue will be resistant to further treatment with associated negative effects on the economics of any process. This papes considers outlets for predominately agricultural and horticultural residues, although by-products from forestry and industry can also affect the economic balance in tern-is of a nationwide lignocellulose strategy.

Upgrading Crop Residues for use as animal feedstuffs

When investigating outlets for crop residues a consideration must be whether their use outside agriculture as a non-feedstuff resource is desirable. Straw, for example is a potentially useful source of energy for the ruminant, although the high lignin content limits its use in this respect. There is about 50% as much energy in the straw, which is usually wasted, as in the seed, which is carefully harvested. However, the availability of energy in straw is low; about 60% is in the form of cellulose and hemicellulose, but a lignin-carbohydrate complex (LCC) exists which renders the nutrients inaccessible to the gut flora of the ruminant. If the bonds in the LCC could be broken or lignin selectively degraded, then straw would present a usef~il energy source for the ruminant as the cellulose would become available. To improve the nutritive value of cereal straws two major feeding constraints of low intake and low digestibility must be overcome.

Techniques employing fungi

Although chcmical and physical mcthods have becn described as ways of upgrading crop bi-producw, thcy can oftcn be expcnsive or cnvironmentally unsound and will no1 bc considcrcd in this papcr. Upgrading lignocellulosic wastes by microorganisms has forincd thc bnsis of a considcrable number of rcsearch programmes. A useful rcview has becn produccd by Arora et al., 1994. Many of the techniques utilize the

Biology International No 35 (August 1997)

waste product as an energy source to produce biomass (e.g mushrooms, meat) and hence to increase the overall protcin yield, while others use this waste energy source to produce chemical feedstock such as alcohol, from the breakdown of cellulose to glucose by ccllulases and the subsequent fermentation of glucose to produce ethanol. These techniques are discussed separatcly later.

Improved digcstibility of lignocellulosic materials requires the breaking of the lignocellulosc bond andlor the selective rcmoval of lignin (Paterson, 1979). 'High tech' microbiological methods require sophisticated equipment, a centralised factory process and again a high cnergy input. However, the processes of most significance to developing countrics (which are of panicular relevance to CAB International) have been those utilising 'on farm' solid state composting techniques using microorganisms naturally occurring on the relevant substrale which, with limitcd environmental manipulation can be maintaincd as the dominant organism during fermentation, without any prc-stcrilisation.

Some investigations havc includcd yeasts (Lekprayoon, 1972) and filamentous fungi (Kahlon and Kalra, 1989). Al1 thesc methods however involve the use of 'high' technology, the substratcs may require considcrablc pre-treatment and the actual degradation is oflcn carricd out in batch o r conlinuous flow fermenters. Conscqucntly the possibility of such processes being economically viable is questionable at the momcnt.

Mcthods requiring 'lowcr' tcchnology have also been assessed. Poole and Smith (1976) used two fungal spcçics growing on loose barley straw hcaps which had been nutritionally adjustcd; onc spccies utilizcd the cellulose while another 'scavenged' excess carbohydrrites. Hcliay and Ptofi (1965) compostcd rice straw supplemented with a nitrogcn sourcc, and thc whole was then pasteurizcd and inoculated with the commcrcially grown mushroom Agaricus bisporus. The resulting compost was termed 'Mycofuttcr' and was said Lo be a suitable protein and carbohydrate source for 'al1 meat, milk, egg and wool producing domcstic animals'. Vcal and Lynch (1984) used a cocktail of' thc nitrogcn fixing bactcrium Clostridiurn butyricum to overcome the high C:N ratio in straw which often rcsults in nitrogen limitation during decomposition and thc ccllulolytic fungus Trichoderma hazianum.

Studics by Kcllcy (1979) and Kcllcy and Scal (1980) idcntificd the possibility of using an organism which occurs naturally on cereal straws to enhance their digcstibility. Coprinus cinereus is known as a wecd organism in mushroom composts whcrc it is an indicator of high lcvcls of ammonia. This observation led to the use of ammonia 10 sclcct for thc growth of C. cinereus in a fermentation process which significantly incrcascd thc digcstibility of whcat straws. This was linked to an on-farm proccss which 'strippcd' aminonia from animal waste for use in thc fermentation proccdure. Othcr workcrs have used urea for this purpose, however the time lag rcquircd for thc urca 10 brcak down and the largcr number of organisms able to utilisc this nilrogcn sourcc lcd to contamination, and the longer fermentation penod mcant that proportionatcly morc cellulose was bcing utiliscd by the fungus and hence digcsti bility dccrcascd.

The low tcchnology approüch has bccn taken up again in rccent years particularly in devcloping countrics whcrc thc wastes from mixcd farming are more readily available on a single sitc (Chawla and Kundu, 1987; Venkatraman et al., 1989). Gupta (1988) uscd C. jïmetarius (cinereus) and urca in a two phase mcthod which has become


known as thc Kamal proccss. Dcvelopments to this technique have moved it towards factory scalc which is no longer suitablc for single farm or small village use.

C. fimetarius has also bccn shown to grow quite easily on straw bales impregnated with a solution of calcium nitrate or a mixture of limestone and ammonium nitrate in solution. The chcmical trcatment is achicvcd by dipping straw bales in the hot solution at 8 0 4 .

Zadrazil and Brunncrt (1981) also investigated Ganoderma applanatum which prcfcrcntially dcgradcs lignin in whcat straw and helps increase the digestibility at lowcr tcinpcraturcs.

The success of thcsc methods Vary. Good degradation and promising bench scale results arc oftcn found, but only limitcd succcss scen when the methods are transfcrrcd to pilot scalc and whcn fccding tnals are carricd out. However, it does sccin that with carcful balance of nutricnts and timing a good quality product can be produccd in a rclativcly short pcriod.

The use of Residues as Substrates for growth of Mushrooms and as Animal Feedstuffs

Mushrooms can makc a signillicant contribution to the human diet. Their protein qualily is midway bctwccn thnt of animal and vcgctable.

Many sinall farmcrs and houscholdcrs in Asia and Africa grow edible mushrooms, oftcn using ccrcal straw as the partial or complcte growth medium. Pleurotus ostreatus can bc grown simply in plastic bags which are stuffed with straw, steamed, and thcn inoculatcd with thc white rot fungus. The residual straw can be fed to ruminants (Bano & Rathnain (1988). In Indonesia, plastic growth bags are used to producc various edible basidiomycctes such as Tramella, Pleurotus, Aurcularia, and Lentinula spccics froin a combination of sawdust from rubbcr trees, nce hullsbran and supplcincnw of othcr nutricnu.

Langer et al. (1982) fcd spent mushroom compost from Agaricus bisporus cultivation to ruminants but found that thc digestibilities were significantly lower than those obscr-vcd for unticalcd whcat straw. This situation is unlikc that of Pleurotus which is an cfficicnl lignin degradcr and hcnce increascs digcsitibility. Zadrazil, (1986) concludcd that thc bcst cdible fungi to give a good yicld of fruiting bodies togcthcr with an incrc:isc in in vitro digcstibility arc Pleurotus spp. and Stropharia rugosuannulata.

Thc succcssful introduction of mushroom growing can provide a high quality nutricnt sourcc for human consumption togcther with an improved feedstuff for ruminants.

Composting

Composting is oftcn fclt to bc simply a way of disposing of wastes, however it should bc undcrstood that thc rcsulting matcrial is a product with a commercial value. The flcxibility of thc composting proccss makes it an attractive proposition for al1 econoinics, as 'wastcs' can bc compostcd on local or farm scale right up Lo large


region widc îücilitics. Whilc the composting process is brought about by a consortium of organisms, fungi play ii significant role in the process.

Most lignoccllulosic malcrials can be compostcd with or without the addition of a nitrogcn sourcc to amcnd the C:N ratio. The resulting quality may be suilable for simple mulchcs and soi1 conditioners or if wcll made can be the equivalcnt of a good quality fertiliser. Composts are known to improve the resistance of crops to palhogcns which rcprcscnts an additional bcncfit of the process (Hoitink & Fahy, 1986).

Composting icchnology can bc dividcd into Reactor and Non-reaclor methods. Non reactor methods arc Iàirly slraighlforward tcchniqucs using windrows or aerated static piles of malcrial. Rcaclor Lcchniques are usually carricd out in concrete or metal containers, mcthods can bc subdividcd into vcrtical flow, horizontal flow or batch tunncl systcms.

Whilc compost tcchnology has bccn undcrstood for many ycars it has bccn neglected in the dcvclopcd world and has only rcccnlly bccn 'rcdiscovcrcd' Vcgctable wastes, wood and bark matcririls which may not bc suitable for upgrading as fcedstuffs are very suitriblc for composting (Smith and Paterson, 1976).

Chernical Feedstocks

Lignoccllulosics can bc uscd ris substrate to producc sourccs of encrgy (alcohol, mcthanc) or as raw inatcrials Tor fermentation to produce fccdstoçk for the chcmicals induslry (acctic acid, acctonc, n.butanol, butancdiol and phenols) (Flickingcr and Tsao, 1978).

Thc potcntial of using isolatcd extracellular fungal enzymes to hydrolyse waste subslratcs has also bccn studicd by a numbcr of workers. These mcthods are usually designed with Lhc intention of rctricving the carbohydratcs produccd for use as chemical fccdstock in othcr proccsscs. Trichoderma viride ccllulases are frequcntly employcd in Lhcsc tcchniqucs to convcrt the ccllulose to glucosc which is in turn fermcntcd Lo Lhc dcsiicd pioduct.

Conclusions

There are many potenti;il outlets for the use of lignocellulosic waste materials. Changing econoiuic factors control which techniques are likely to be viable at any one time. However, if there is a commitment to develop a national strategy many of the outlets addressed in this paper can be used in parallel to produce a viable programme. In the UK, waste lignocellulosics were not considered to have any ptirticular value until 1993 when the burning of straw in the field became illegal. In anticipation of this niove in 1992 The Straw Utilisation Association was launched. The füct that the U K now has a society concerned solely with this topic suggests that straw niay have left the realm of problem waste in the UK and has entered that of resource. Similarly on 1st October 1996 new laws came into force which imposed a tax on a11 materials disposed of in landfill sites. This has generated immediate interest in composting of wastes which has become something of a


growth industry iri the UK. The selection of method, substrate, technology and implementation therefore depends on the local socio-economic conditions.

References

Arora, J.K., Kakkar, V.K. and Sukhvir, Kaur (1994). Bioconversion of agro-residues for food and fced. Annuul Review, Agriculiural Review 15(4), 205-217

Bano, 2. and Rathnam, R. (1988). Mushroom Newslefter for lhe Tropic 6 , 11. Chawla, A. and Kundu, S.S. (1987). Chcmical composition and dry matter disappearance of

mould coloniscd whcat suaw. Farm Animals 2,70-73. Flickingcr, M.C. and Tsao, G.T. (1978). Fermentation substrates for cellulosic materials.

Annuul Reporu on Fernienlafion Processes 2,23-4 1 . Gupta, B.N. (1988). Dcvclopincnt and concept of karnal process for fungal ueatment of

ccrcal straws pp.46. Procccding of International Workshop on fibrous crop residues as aniinal Sccds. NDRI (SRS) Bangalore, India.

Hcltay, 1. and Pctofi, S. (1965). Mycofuttcr, Mushroom Science 6, 287-296. Hoitink, H.A. and Fahy, P.C. (1986). Basis for the conuol of soilborne plant pathogcns with

coinposts. Ann. Rev. Phylopathol. 24,93-114. Kahlon, S.S. and Kalra, K.L. (1989). Biological upgradation of wheat suaw into protein-rich

fccd. Journal of Reseurch, Punjab Agricultural Universify 26,257-265. Kcllcy, J. (1979). Ecological studics on the microbial upgrading of suaw, Ph.D. Thesis,

Univcrsity OS Aston, Biriningharn. Kcllcy, J. and Scal, K.J. (1980). Thc cffecu of addcd niuogcn on the microflora of waste

suaw. In: Biode!eriorulion, cds. T.H. Oxley, D. Allsopp and G. Bccker. Pilrnan Publishing London.

Langcr, P.N., Schgal, J.P., Rana, V.K. Singh Marmohan and Garcha, H.S. (1982). Utilization of Aguricus bi.sj~oru.s - culiivatcd spcnt whcat suaws in Lhc ruminant diet. Indian Journal of Animal Science 52, 634.

Lckprayoon, C. (1972). Production of Candida ufilis from rye-grâss straw hydrolysate. M.S. Thcsis, Orcgon Staic Univcrsity.

Palcrson, R.R.M (1979). PhD Thcsis, University of Manchester. White rot fungi and their erfcct on barlcy straw.

Poolc, N.J. and Sinith, A.L. (1976). Thc potcntial of low technology. Octagon Papers 3. Ccllulosic subsuatcs. Univcrsity of Manchcstcr.

Smith, J.E. and Paicrson, R.R.M. (1976). Trcc bark - a usable commodity. Process Biochemislry, July/August, 4 1-48.

Vcal, D.A. and Lynch, J.M. (1984). Associatcd cellulolysis and dinitrogen fixation by co- culturcs ol' Trichudermu hurzianum and Closlridium bufyricum. Nature 310 (5979), 695- 697.

Vcnkavainan, K., Kishnn Singh, and Gupta, B.N. (1989). Changes in composition of straws on Lrcatiiicni with Collybiu velufipes and Coprinus fimefarius. Indian Journal of Animal Nulrilion 6, 307-316.

Zadrazil and Brunncrt (1981). Invcsligation or physical paramctcrs important for the solid statc fcrincniütion of suaw by white rot fungi. European Journal of Applied Microbial Biolcchnology 1 1, 183

Zadrazil, F. (1986). Whitc rot fungi and mushrooins grown on ccreal suaws: aim of the proccss, final products, scopc for Lhc future. pp.55. Procecding of Workshop on degradalion of lignoccllulosic in ruminants and in industrial processes. Lelystad, Nclherlands.

Biology lnternational No 35 (August 1997)

Biosystematics Meeting the Demand

David L. Hawksworth President, lnternational Union of Biological Sciences

Director, lnternational Mycological Institute, Egham, Surrey TW20 9TY, U K

I n t r o d u c t i o n

Biosystematics, or biological systematics, is the part of science that embraces the taxonomy and nomenclature of living and fossil organisms and elucidates their evolutionary relationships. It provides the communications system of biology, is at the heart of al1 discussions on biodiversity, and is the key component ingredient of biodiversity assessment and monitoring strategies.

A suite of international initiatives is underway to address different aspects of the biosystematics conîponents of biodiversity programmes at al1 levels, and several of these are incorporated into the conceptual framework of DIVERSITAS. The International Union of Biological Sciences (IUBS) has been concerned at the mismatch between the needs for and level of resourcing accorded to biosystematics world-wide for many years, and has contributed to several of the initiatives now underway or seeking external funding.

In order to increase awareness of the needs for biosystematics, IUBS, with the support of the U K Systenîatics Association, convened a symposium during the Fifth International Congress of Systematic and Evolutionary Biology in Budapest on 21 August 1996 on Biosystematics: Meeting the Demand. Four of the papers from that meeting follow in this issue of Biology International. The meeting was chaired by Professor Vernon H. Heywood, and other contri-butors included Professor D.H. Janzen and Professor R.R. Colwell.

Recognizing that non-specialists might not readily appreciate the differences in the foci of the severül related global initiatives in biosystematics, IUBS also arranged a meeting of the leaders of the various programmes and officers of pertinent organizations at the International Mycological Institute on 23-24 March 1997. The meeting was chaired by Professor Sir Ghillean Prance on the first day and myself on the second. The planned output was a text explaining how the current series of nîultidisciplinary çonîplementary initiatives cooperate in addressing vanous aspects of world needs in biosystematics and taxonomy. This agreed text, and accompanying diagrammatic representation (Fig. 1) which are also being made available separately, constitute the remainder of this article.

Note that the following text does not inçlude numerous initiatives and organizations concerned with particular g~-oups of organisms or which are national or regional in scope.

Biology Internationa/ No 35 (August 1997)

The Initiatives

BioNET-INTERNA TIONAL (BI) Building capacity

An intergovernmental global network of people and institutions for capacity building in trixonomy in the developing world. It is a series of inter-linked LOOPs (Locally Orgariized and Operated Partnerships) owned and managed by the governments that establish them, to pool existing resources and facilitate the transfer of taxonomic skills, technologies and information from the major world centres to developing country institutions. The groups of organisms varies with LOOPs but always include the most species-rich groups where developing country capabilities are poorest. LOOPs exist in the Caribbean, East, West and Southern Africa, South-East Asia, and the South Pacific. BI was initiated through CAB INTERNATIONAL and is endorsed by FAO, UNDP, UNEP, UNESCO, the Secretariat to the Convention on Biological Diversity, and bilateral and multilateral development agencies; it is now activated in some 120 countries.

Contact: Fax: (+44)[0]1784 470909; E-mail: [email protected]

SPECIES 2000: Iiidexing the World's Known Species Accessing species information

The objectives of Species 2000 are: (1) a catalogue of the world's known organisms; and (2) a framework for linking species databases world-wide. These are created by common access to an array of taxonomic databases dispersed at many sites on the Internet. Species 2000 services will provide species catalogues of al1 known species, with verification of taxonomic details, and linkages to databases on species, collections and germplasm in many countries. Initiated by IUBS, CODATA and IUMS, endorsed by UNEP, and participating in the Clearing House Mechanism of the Convention on Biodiversity.

Contact: Fax: (+44)[0] 1703 594434; E-mail: [email protected]

Systeïnatics Agenda 2000 International (SA 2000/I) Exploring species diversity

Systematics Agenda 2000 International (SA2000/I), the systematics component of DIVERSITAS, has three broad missions: to discover, describe and inventory global species diversity, to analyze and synthesize the information from this global discovery effort into a predictive classification that reflects the history of life; and to organize this infonnation in an efficiently retrievable form that best fits the needs of science and society. Within this context SA2000fl will promote national, regional, and international efforts to build systematics science through inventories, phylogenetic research, and the creation and dissemination of the systematic knowledge bases.

Contact: Fax: (+3 1) 71 527 351 1; E-mail: baas@rulrhb. LeidenUniv.nl; or Fax: (+ 1) 212 769 5759; E-mail: [email protected]

Interizational Organizaîion for Systernatic and Evolutionary Biology (IOSEB) Providing scientific liaison

Established to promote the development and awareness of the subject, it organizes the International Congress of Systematic and Evolutionary Biology (ICSEB), provides a communication mechanism between pertinent national and regional societies, and promotes the development and awareness of biosystematics and


evolutioniiry biology. Coiistitutes the Seçtiori for Systen~ütic and Evolutionüry Biology within IUBS.

Contact: Fax: (+36) 1 175 9539; E-mail: [email protected]

THE TASKS (1)

Ijuilding capacity f Accessiiig species jtlxploring species i ~ r o v i d i n ~ scientilïc (2) i iiihriiiation j diversity j liaison

Species 2000 IQSEB

(1) This dirigruii~ is dcsigiicd lo slrcss llic inain l'ocus ol' ihc diffcrciil inilialivcs in rclalioii lo ~ h c kcy Lasks; ull will or Iiavc tlic polcn~ial to conlributc ~o paris of al1 iasks.

(2) Building capacily i i i dcvcloping counlrics by ~cclinical and profcssional training, rchabililalion of syslcino~ics facililics and dcvclopiiicnl of idcnlificalion lools.

(3) Discovcriiig, dcscribiiig and invciiloryiiig global spccics divcrsiiy, analyzing and synlhcsizing lliis information iiilo a prcdiclivc classificalion, and organizing lhis in an cfficicnlly rctricvoblc forin.

The Sponsoring Organizations

LIIVERSITAS An inlcrnalioniil progi'nmmc iliid conccplual Sramcwork Sor al1 aspccls of biodivcrsily sciciicc, cs~iiblislicd in 1991. IL is r i coiisorliuin of IUBS, SCOPE, UNESCO, ICSU, IGBP-GCTE, and IUMS. DIVERSITAS, as an uinbrclla, is lhc only cxisling prograinrnc lhal coordiiialcs il broad rcscarcli cfIoi-L in ihc biodivcrsiiy scicnccs al Llic global lcvcl. I L is uniquc rlirougli ils fuiiclionally liiikcd Elcincnis and Spccial Targcl Arcas of Rcsciirch (STARS).


ICSU The Intemalional Council of Scientific Unions, established in 1931, 10 promote international scicnlific aclivity in the different branches of science and their applicalions for thc bcncfit of humanity. It comprises 95 scientific academies or research councils and 25 Scientific Unions ( t g . IUBS, IUMS), international disciplinary organizalions. ICSU also establishes commiltees for specific purposes (e.g. CODATA, SCOPE).

IUBS The Intcmalional Union of Biological Sciences, founded in 1919, is a Union of 83 intemalional socictics and othcr organizalions and 41 scicnlific academies.

IUMS Thc Intcrnrilioniil Union of Microbiological Sociclics. established in 1980, is a Union of national socictics conccrncd wilh microbiology.

CODA TA The Commiilcc on Data for Sçicnce and Technology, eslablishcd in 1966, working on an inlcrdisciplinary bcisis, sccks 10 improve the quality, reliabilily, processing, managcmcnl and acccssibilily of data of importance to scicnce and technology in al1 the disciplines covcrcd by ICSU.

Thc Scicnlific Coinmillcc on Problcms of thc Environment, founded in 1969, which assemblcs, rcvicws and asscsscs the available knowlcdge on environmental problems. An ICSU commillcc.

BioNET-INTEKNATIONAZ2 Consultative Croup (BZCG) A group of cmincni scicntisis from the devcloping country subregions, the major taxonomic instiiutions of lhc dcvelopcd world, and reprcsentalives of international devclopmcnt agcncics who launched BI ihrough CAB INTERNATIONAL in July 1993 and who promotc, fostcr and manage this global network through the Coordinaling Commiticc (BICC) and the Technical Secretariat (TECSEC). Activities have bccn sponsorcd by thc BI FUND and vanous development agencies.

CAB Irlterrlatiorzal An inlcrgovcrnmcnial organization cstablishcd in 1929 dcdicated to improving human wclfrirc through 1hc disscmination, application and gencraiion of scientific knowlcdgc, wilh p;irlicul:rr allcnlion 10 thc nccds of dcveloping counlries.

Acknowledgements

1 wish 10 acknowlcdgc LIIC conlribulions of IUBS and the Sys~cmalics Association to Lhc cxpcnscs of spcnkci-s ai ~ h c Budapcsl symposium. to IUBS for travel cosls of initialivc rcprcscnlalivcs al ~ h c Egham mccling, and parlicularly 10 the chairs and participanls al bolh occasions.


The Perspective of the Systematist

Bernard R. Baum* Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre,

Neatby Building, B2, Central Experimental Farm, Ottawa, Ontario K1A OC6, Canada

The systematist today is facing a problem unparalleled since the beginning of the profession. The problem of resources and funding has always existed as we shall see below. The new problem is that systematics as we know it is undergoing a major charige. Inyfact most of the discipline of systematics has been taken over, or has been absorbed-,by other disciplines which in fact practice it, sometimes inadequately, withoht mention of the word "systematics" at all. Although my presentation is from the perspective of a plant systematist, what follows might apply to systematists other than those of plants.

Past and Present with Emphasis on Funding

Aside from their use as food, the study of plants for medicinal purposes attracted the interest of the Greeks and later Europeans during the Middle Ages. Those studies were reflected in the herbals of which the most famous were those written by Theophrastus, Dioscorides, Bunfels, Bock and Fuchs. In those herbals the plants were assembled together according to their practical use, i.e. into special purpose classifications.

The urge to discover new sources of spices and medicinal plants came along with the general wave of mounting long voyages to discover unknown lands for yet undiscovered riches. These long voyages were often initiated by royalty who had an economic interest in new crops, medicinals and spices. Typical examples are Marco Polo to the Far East and Columbus to the Americas. It is through these voyages that biodiversity on the globe gradually became recognized. At about the same time the von Guttenberg method of printing took off and thus allowing the publication and dissemination of the herbals. Together, these two events, voyages of discovery and the dissemination of printing, triggered a sudden increase in interest in plant identification, in the search for medicinal plants, and finally in the founding of chairs of botany at European medical schools. The first chair of botany was founded in Padua, Itüly in 1533 (Mriyr, 1982).

The science of systematics was borne out of the necessity to arrange the accumulated knowledge on plants for identification purposes for the very practical end of knowing and using medicinal plants.

* Thc opinions exprcsscd in this paper are striclly m y own and do not reflect those hcld by Agriculture and AgriFood Canada, nor do they purport to reflect necessarily those hcld by sysïematisLs.


A great step in classification was made by Cesalpino in his major work De Plantis (Cesalpino, 1583). This book influenced botany for the next 200 years. Another innovation credited to Luca Ghini (1490-1556) was the technique of pressing and drying plants, which we as systematists take so much for granted today, and which laid the basis for herbarium collections. Luca Ghini also established the first university botanic garden in Pisa, Italy, in 1543 (Mayr, 1982). Thus, the herbarium as a tool for botanical research dates back to the 16th century.

Since the 16th century different kinds of people were motivated to invest time and resources in the pursuit of systematics. Clencs, for instance, thought that they could better understand the master plan in their religious beliefs. This is true for the French and British missionaries who collected in the 19th century. Medically oriented people were genuinely interested in the search for healing compounds, e.g. Asa Gray (1 81 0-1 888). With the discoveries of many new species came a need to arrange and catalogiie that knowledge. It was Linnaeus (1707-1778) who laid the foundation of binomial nomenclature (Linnaeus, 1753) that enabled the cataloguing of the increasing ktiowledge of biodiversity. Binomial nomenclature continues to be practised and is the corner stone of our modern nomenclature. It was through the continuing support of royalty and a growing number of aristocrats, however, that collecting expeditiotis were mounted and supported, for instance the famous expeditions of Captain Cook. The basis for the major museum collections of today was laid by botanical explorers with the support of wealthy benefactors, e.g. Sir Joseph Banks (England, 1743- 1820), George Clifford (Holland, 1685-1760), Kunth (Germany, 1788-1850) and others. Serious and wealthy amateurs, such as Boissier (Switzerland, 18 10-1 885) were able to finance their own voyages and contributed greatly to the major museum collections. Other dedicated amateur naturalists or professional botanists, not rich at all, were so devoted to the pursuit of botanical knowledge thrit to support themselves they sold duplicated collections, or just ended in extreme poverty, such as Kotschy (1 813-1866) and Rafinesque (1783- 1840).

The knowledge about biodiversity that has accumulated in the last 250 years was made possible by committed amateur naturalists and dedicated scientists through privately funded sources or by their own meagre means. Government agencies have only in past few decades dedicated a certain amount of funding to systematic botany due to the perceived need for developing knowledge about the biota as part of our custodianship of natural resources and for conservation and protection programs. I t is ciirrently perceived that in order to make informed decisions about Our genetic resources one must study them first for the (inter-)national good. To date the following statistics are available (Baum, 1996): 1023 gene banks conserve seed material, 1300 botanic gardens fulfil various recreational and professional activities, and the collections in 2638 herbaria are indispensable for the continuous scientific activities in systematics. Most of these gene banks, botanic gardens and herbaria sire currently funded by national or municipal agencies, some by international agencies, others by universities. A great number of botanical gardens are funded by private organizations. The institutions responsible for these collections are currently experiencing a drastic shortage of funds threatening their continued existence, including the research staff with the capability to use these collections.


The Transformation of Systematics

Developmental periods of systematics and its major landmarks were outlined by Alston & Turner (1963) and others. Constance (1964) has eloquently summarized the waves of new technologies until that date, the influence of each on systematics, and the impact each technology had on the transformation of systematics. 1 will very briefly highlight a few instances of recent transformations.

Since the tiirn of this century systematics has developed beyond the science of collecting and cataloguing. Jaccard (1908) developed a measure of resemblance based on mathematical principles. Although he was concerned with plant distribution, his coefficient of resemblance later became used in numerical taxonomy and numerical ecology as well as in other disciplines concerned with numerical comparisons. Pearson (1926) developed his coefficient of racial likeness, and Fisher (1936) was concerned with discrimination between individuals or taxa based on multiple measurements, i.e. characters. Most systematists did not take up the mathematically based methods for their comparative studies until the 1960s, when the increasing use of computers played a major role in façilitating the storage and manipulation of data. In the meantirne, geneticists used numerical methods increasingly since Mendel, and have taken up Fisher's and Pearson's work, which in fact addressed taxonomic problems, along with other mathematical contributors. Today computer data handling and mathematically based algorithms are absolutely essential to modern systematics.

Other disciplines appeared. Cytology and cytogenetics became the disciplines of choice for studying genome relationships of our major crops and their wild relatives. This helped planning germplasm enhancement strategies in a number of plant groups. Cytogeiietics perhaps started with the work of Rosen-berg (1909) on Droseru, and continues until today using for the most part molecular techniques (see Jauhar, 1996). Classical cytology and cytogenetics, investigating chromosomal numbers and behaviour i n artificial crosses, was indeed taken up by systematists. Thus systematics became transformed into an experimental science, i.e. in addition to morphological examination of her-barium material, live material was used for cytological investigation as well as n~orphological examination, and with additional emphasis on statistics and population genetics. This transformation of systematics became known as biosystematics, an interdisciplinary group calling themselves The Biosystematists being founded in the US in 1937 (Smocovitis, 1994); the term "biosystematics" is generally regarded as coined by Camp & Gilly (1943) [as "biosystematy"] (see also Camp, 1951).

Chemical and biochemical investigations with some relevance to systematics date back to the 19th century. For instance, according to Hegnauer (1958), A.P. de Candolle (1778-1 841) was concerned with correlating chemical compounds with medical properties with morphological features of species, genera and families. Although most systematists were not concerned with chemistry, research into the chemical properties of plants was continued by chemists. From these investigations by chemists, the relevant work wiih taxonomic implication was compiled and reviewed by Alston & Turner (1963) "as an effort to consider perspectives in biochemical systemritics". This marks the beginning of biochemical systematics. Many investigations have been carried out since then, primarily on micro-molecules


and especially on secondary metabolites, summarized for the period until 1982 by Harborne & Turner (1984).

In addition to using micro-molecules as tools, systematists have increasingly engaged in using macro-molecules as the techniques for their analysis became widespread and relatively affordable. Protein variation has been analysed through serological coniparisons (Fairbrothers, 1977), allozyme electrophore-sis (Crawford, 1990), and the laborious anlino acid sequencing (Boulter, 1980).

The advent of two new sets of tools brings us to current times, i.e. DNA systematics. These tools are restriction endonuclease analysis and DNA sequencing. Although prohibitively expensive at the time, DNA sequencing had been recognized as early as 1965 as a most powerful tool for evolutionary analysis (Zuckerkandl & Pauling, 1965). DNA sequencing, as well as some less recent tools, is most ofteii used by non systenlatists to tackle problems of a systematics nature. For instance, the chapter on "Repeated DNA sequences and polyploidy in cereal crops" (Mitra & Bhatia, 1986), and the study using repeated nucleotide sequences to study the relationships among polyploid species of Triticum (Dvorak & Dubcovsky, 1996) are examples of taxonomic problems that have been tackled by non proclaimed systematists. The molecular analysis of the U genome in wild wheats (Chee et ul., 1995), the plasmon analysis in wheat and allied Aegilops (Tsunewaki, 1996) and the work on genetic diversity of inbred lines of maize using protein markers (Burstin et al., 1994), for example, al1 use numerical taxonomic methods of analysis, such ris principal coniponent analysis, cluster analysis, and cladistics.

1 stated that biotechnologists often investigate problems of a biosystematic nature. Conversely, however, systematists that are using modern tools such as DNA sequencing or RAPDs can be construed as non-systematists, simply because of their choice of tools, overlooking their contributions to resolving taxonomic problems which include the building of reference systems, including nomenclature. A new interdisciplinary science has evolved due the utilization of the powerful methods of recombinant DNA technology. This new discipline has been called "DNA systematics" (Dutta, 1986). Included in it are a suites of nucleotide (DNA and RNA) technologies, such as DNA finger-printing and typing, DNA sequencing, evolutioriüry inference in specific genes and gene families, restriction enzyme analysis, e.g. chloroplast DNA, amplification of DNA using the polymerüse chain reaction. Terms with broader and different connotations than DNA systematics, such as n~olecular systematics and evolutionary biology, often overlap with i t i n practice. Molecular systematics also includes isozyme analysis and other approaches. Evolutionary biology, is primarily concerned with patterns and processes, and incorporates DNA systematic methods and data. The terms are often used interchangeably although DNA systematics is concerned with both classificatory and evolutionary problems.

What is happening today is that non-systematists are in fact working as practising systematics, i.e. using taxonomic methodology of classification and phylogenetic analysis, but failing to carry out the work through to forma1 taxonomies that can be used as reference systenis with appropriate nomenclature. Conversely, systematists are increasingly turning to modern tools, such as DNA sequencing, that became

Biology International ND 35 (August 1997)

available through vririous efforts to solve other practical needs. Systematics is thus, in a sense thriving, as it is transfomied into molecular biology, or other disciplines that are in fact prrictising systematics, whereas systematics in its traditional sense is in trouble. However this blending of systematics does often mean that it superficially appears to be in decline in its discipline or its own right. But systematics has as one of its aims to provide a convenient method of identification and commuiiic;ttion (Davis & Heywood 1963). This requires the formalization of classes and trixononiic systenis with appropriate nomenclature to achieve the aim of comnlunication, rind appropriate means for the identification of the classes established by the research using the modern tools. In other words, some aspects of the conventional systematics must also be taken up by DNA systematists and others who investigate problems of systematic nature.

The Demand

Public institutions were erected as a result of the support of governments for scientific investigations for the public good. Now systematists have become victims of shortages of funding, rzlong with scientists in other disciplines, due to governnients cutbacks riiid downsizing.

The demrind is now clearly for DNA systematic work, i.e. DNA systematics. It is imperritive thrit systeniatists use the new tools to make their work relevant, and to be recognized as iieeded to achieve the objectives. But unfortunately, the term "systematics" is currently unpopular. Thus the demand is for research using recombinant DNA technology and associated tools irrespective of the name of the profession. The terni "systematist" or "taxonomist", often insinuates the picture of a traditional taxonomist working in isolation in an herbarium, divorced from practical problems and often changing the names of the plants. To survive as a professional the systematist must denionstrate not only that helshe is able to use the same modern tools of sesearch that are used by others, but in addition that helshe relates the DNA findings to the iîiorphology of the plants, i.e. their correct identity, to produce iiseful identification and classification systenis. 1 indicated that systematic tools rire now used as a matter of routine by investigators in various other disciplines. Using the tools alone does not ensure the correct identity and classification of the material. The DNA systematist can provide this, and is expected to do so.

Many of the theoretical advances have been made on mode1 systems that were also of practical importrince, such as wheat, rice and maize. Much of the research funding can be obtained, usually from private industry, but only if it can be demoiistrated to be of imiiiediate practical importance or that there is a market need for it. Meeting these denxtnds is not an easy task but probably never was because sponsors were ofteri not erisy to find in the past. Balancing these demands, both new and curseiit, ren1;iins a fundrimental challenge for biosystematists in the present and future.

Acknowledgements

1 am graicful to Drs D.A. Johnson (Univcrsiiy of Ottawa) and P.M. Catling (Eastern Ccrcal and Oilsccd Rcscaich Ccnlrc) for commcnting on an early draft of this paper.


References

Alston, R.E. and Turner, B.L. (1963) Biochemical Syslematics. Englewood Cliffs, N.J.: Prenticc-Hall.

Baum, B.R. (1996) Statistical adcquacy of plant collections. In Sampling the Green World (T. Stuessy and S. Sohmcr, eds): 43-73. Columbia Univ. Press.

Boulter, D. (1980) Thc use of amino acid sequence data in phylogenetic studies with special refercncc to plant proteins. In: Chemosystema~ics, Principles and Practice. (F.A. Bisby, J.G. Vaughan, and C.A. Wright, eds): 235-240. London: Academic Press.

Burstin, J., dc Vicnnc, D., Dubrcuil, P. and Damerval, C. (1994) Molecular markers and protcin qualilies as gcnetic descriptors in maize. 1. Genetic diversity among 2 1 inbred lincs. 7'heoretical and Applied Genefics 89: 943-950.

Camp, W.H. (1951) Biosyslcmaty. Brittonia 7: 113-127. Camp, W.H. and Gilly, C.L. (1943) Thc structure and origin of species. Brittonia 4: 323-385. Cesalpino, A. (1583) De Planfis Libri XVI. Florcnce. Chee, P.W., Lavin, M. and Talbcrt, L.E. (1995) Molecular analysis of evolutionary patterns in

U gcnomc wild whcats. Cenome 38: 290-297. Constance, L. 1964. Systcmatic bolany - an uncnding synthesis. Taxon 13: 257-273. Crawford, D.J. (1990) Plunr Molecular Systemurrcs. Mucro Molecular Approaches. John Wiley

& Sons, Ncw York. Davis, P.H. and Hcywood, V.H. (1963) Principles of Angiosperm Taxonomy. Edinburgh: Olive

& Boyd. Dutta, S.K. (1986) Inlroduction. In DNA Sysfemufics (S.K. Dutta, ed.): 1: 3-4. Boca Raton:

CRC Prcss. Dvorak, J. and Dubcovsky, J. (1996) Gcnome analysis of polyploid species employing

variation in rcpcatcd nuclcolide sequenccs. In: Methods of gcnome analysis in plants (P.P. Jauhar, cd.): 133-145. Boca Raton: CRC Press.

Fairbro~hcrs, D.E. (1977) Pcrspcctivcs in plant scrotaxonomy. Annals of the Missouri Botanical Garden 64: 147- 160.

Fisher, R.A. (1936) Thc use of multiple mcasurcmcnts in taxonomie problems. Annals of Eugenics 7: 79 188.

Harbornc, J.B. and Turncr, B.L. (1984) Planr Chcmosysfemafics. London: Acadcmic Press. Hcgnaucr, R. (1958) Chcinolaxonoinischc Bctrachlungcn. Pharmacia Acfa 1-lelvefiae 33: 287-

305. Jaccard, P. (1908) Nouvclles rcchcrchcs sur la distribution florale. Bullefin de le Société

Vaudoise des Sciences Nu rurelles 44: 223-270. Linnaeus, C. (1753) Species Plunfarum. Stockholm. Mayr, E. (1982) The Growth of Biological 7'hought. Cambridge, Mass.: Harvard Univ. Press. Miua, R. and Bhatia, C.R. (1986) Rcpeatcd DNA sequcnces and polyploidy in cereal crops.

In: DNA Sysfemufics (B.K. Duua, cd.): 21-43. Boca Ralon: CRC Press. Pearson, K. (1926) On tlic cocfficicn~ of racial likcness. Biomefrika 18: 105-117. Rosenbcrg, O. (1909) Cytologische und morphologische Studien an Drosera longifolia x

rofundifoliu, Kunglige Svcnska Vefen.skupsucudemiens llandlinger 43: 1-64. Tsuncwaki, K . (1996). Plasinon analysis as Lhc countcrparl of gcnome analysis. In Methods of

Genome Anuly.sis in Plunrs (P.P. Jauhar, ed.) 271-299. Boca Raton: CRC Press. Zuckcrkandl, E. and Pauling, L. (1965) Molcculcs as documcnts of cvolutionary history.

Journul of Theorcricul Biology 8: 357-366.


The Perspective of the Biodiversity Prospector

H.G. Wildman AMRAD Discovery Technologies Pty. Ltd, 576 Swan Street, Richmond,

Victoria 31 21, Australia

Introduction

Our relative ignorance of biodiversity and its genetic makeup is highlighted by the fact that, while the total niimber of plant, animal and other species thought to be in existence ranges from 3-30 million, only 1.5 million have as yet been descnbed taxonomically (May, 1992).

Large numbers of new species of organisms remain to be discovered, parti-cularly in tropical regions and in the world's oceans. However, habitat loss and destruction and the accoinpanying species loss will include some species new to science which have the ability to produce important, but as yet undiscovered, chemical molecules. As such, many companies and institutes are accessing the world's biodiversity with increased vigour and prospecting for new chemical entities such as natural products, fine chemicals and enzymes, and for new biological entities to act as biocontrol and bioremediation agents. 1 would like to present a view of biosystematics needs from the perspective of a biodiversity prospector searching for novel natural products of pharmaceutical importance.

Changes in Prospecting for Novel Natural Products

Until relatively recently, most major pharmaceutical companies have operated screening programmes with a predominantly bacterial (and especially fila-mentous bacteria, such as streptomycetes) and fungal focus. These screening programmes have usually been of a high throughput nature, involving the isolation and screening of many thousands of microorganisms per year. To that end, isolation conditions have been developed and employed to both selectively target groups of microorganisms (e.g. Long & Wildman, 1993) and to selectively eliminate common "weedy" org~inisins (e.g. Wildman, 1991). In addition, many of these isolation programmes have tended to focus on microbes from soi1 samples, despite the well recognised limitations of this approach with respect to the diversity of microbes isolated (e.g. Bills & Polishook, 1994) .

With the increased difficulty in discovering new medicines - viz. in 1961, over 90 new medicines were launched, but less than 50 were launched in 1980, and just over 40 in each of the past few years - there has been a change of emphasis within the natural products discovery units of pharma-ceutical companies from searching for novel iiatural products that could be delivered to market, to searching for novel lead compo~inds. These lead com-pounds would eventually find their


way to iiiarket after cheiiiical iiiodificatioii andlos a coniplete cheiiiical synthesis beiiig devised.

This somewti;it "pliilosophicril" change to serirching for riew natural products has opened up opportiinities for screeiiiiig niany other organisnis for natural product production. Larger companies have expanded into screening organisms of differeiit types to tliose tliey Iiad been screening for nirtiiy yerirs, and snialler compaiiies have started up also to screeii these organisiiis or to provide the Irirger coiiipaiiies with these orgariisnis or their extracts for screening.

Anotlier receiit cliaiige to screeiiing practice fias been tlie advent of the extract library to divorce [lie previous temporal liiik between extract production and extract screeiiing (Fig. 1). The nbility to seprirate the rüte of extract productioii froni the rate of screeiiiiig extncts (provided, of course, tlint there are more extracts in storage at any one tinie ttinii :iriy orle screen would waiit to test), and the ability to rescreen already preprired extrncts as iiew screeiis conie on-line, has mennt that for the first tinle p1inriii;iceutical cotiipniiies are begiriiiiiig to questioii how nxiny extracts they sliould have and of wti;it coiiiposition they should be with respect to the types of organisms represerited.

Figure 1. Tlie psiricipal of an extrrict librriry to separrite extract production froni extrrict screeiiiiig.

What have these changes in the interests of companies screening for natural products meant to biosystematists?

Screening 0 Production

In the past maiiy coi1ip;inies screened snniples often sent i n willy-tiilly by collectors, soiiie of wlioin siriiply labelled their subniissioiis "genus species uiiknowii". Tliese efforts were often "barely better tliari worthless" (R. Doiiovick of tlie Natiiinl Prodiicts Brriiich of the N:itioiial Cancer Iiistitiite, USA as cluoted by Booth, 1987).

Extract Library


Routine species identification can be a problem for the biodiversity pros-pector, because of a lack of appropriate tools or keys to assist with identification, and especially the rapid identification required for the often large numbers of organisms processed. As Alberch (1993) has noted, the greatest problems will be encountered with specimens collected in areas where there is a high proportion of new species. The quality of these collections will be dependent upon the accuracy of their species identifications. As such, it has been recognised that new taxonomic tools and techniques will be required to support biodiversity studies (Alberch, 1993; Krebs, 1992; Systeniatics Agenda 2000, 1994) and the same can be said of bioprospecting.

Plants

Few pharmaceutical companies were screening vascular plants in any great nun-ibers in the 1980s, but within 10 years many larger and a number of smaller companies were actively accessing plants for screening. Accompanying this change hüs been an increase in the use of the taxonomic expertise of the world's botanic gardens, and in collaborütions between industry, gover-nment institutes such as the USA'S National Cancer Institute, and botanic gardens. Botanic gardens also often rict as cleariiig houses or "middle men" between suppliers in less developed countries and western companies interested in screening their botanic san-iples. The existence of many established botanic gardens and the training of botanists for positions within the botanic, horticultural and forestry industries has enabled the increased interest in screening botanical samples and its subsequent biosystematic demands to be largely met. Undoubtedly, however, the continuing search for and discovery of new plant species will put increasing pressure on these existing biosyst-ematic resources.

With the incrensed interest of pharmaceutical companies in screening "unusual" microorganisms for the production of novel metabolites, it has become increasingly difficult to usefully use the existing commercial (e.g. APITM, BIOLOG'rM) or other systems (e.g. Williams et al., 1983) that have been developed in the past for rapid microbial identification or for identification of large numbers of isolates. These systems have been developed generally for clinical use or for use with coniniorily encountered environmental samples, and thus have proved to be difficult to use or of little value for the taxonomy of more unusual microbes. For example, having to n~odify these systems to allow growth of, for example, thermophiles, acidophiles or marine organisms, may be difficult and results are generally not applicable to the existing databases developed for these systems.

Modern molecular systematic methods have quantified what many bioprospectors intuitively knew; that is, that a huge untapped biodiversity of micro-organisms existed. The continued screening of microorganisms for natural products, the developn~ent of novel methods for their isolation, and the screening of new habitats in the search for novel n-iicrobes has created demands for rapid and sensitive taxonomie methods (Table 1) together with high quality databases. Molecular systeinatic nietliods are fulfilling these deniaiids.


Table 1. Taxonomie methods available for rnicrobial isolates (After Review of UK Microbial Culture Collections, 1994).

Molecular systematics methods offer levels of characterisation that were not dreamed of 15 years ago. Although organisms can be characterised at both coarse (viz. genus) and fine (viz. species, strain) levels, pharmaceutical bioprospectors have to decide at what stage they apply these techniques to remove sample redundancy. That is, before or after screening the organisms? For example, the costs in both time and money in utilising molecular methods to characterise microbes to certain levels before screening need to be compared with the costs and effort in high throughput screening followed by dereplication after screening.

TYPE OF METHOD

P h e n o t y p i c

Numerical taxonomy

Morphological study Rapid enzyme tests

C h e m o t a x o n o n i y

P r o t e i n s

Fatty acids Polar lipids Quinoncs Sugars Amino acids Nucleotide contenls (G+C) Whole orgünism 'f ingerpriri t ir ig '

Nucleic acid ( m o l e c u l a r )

DNA 'fingerprinting'

Sequence data comparisons

Molecular probcs

METHODOLOGY

Observed characteristics of organisri

Classification by computation of many phenotypic t r a i t s Light and elcctron microscopy Chrornogenic or fluorogenic tests

Comparison of component molecules of cells

Ce11 'fingerprints', enzyme properties, immunology Gas chromatography (GC) Thin Layer Chromatography (TLC) High Pcrforrnance Liquid Chromatography (HPLC) TLC, GC, Mass Spectroscopy (MS) TLC, HPLC HPLC Curie-point pyrolysis, MS, Infra Red (IR) & Raman spec t roscopy

Comparing gene and other nucleic acid sequences

Plasmid profiling, ribotyping, (Random Amplified Polymorphic DNA (RAPD's), Restriction Fragment Length Polymorphisms (RFLP's), Amplified Fragmcnt Lcngth Pol ymorphisms (AFLP's) 5 s RNA. 16s RNA, 18s RNA, 23s RNA, 28s RNA, spaccr regions Locating taxon-specific radioactive or fluorescent p r o b e s


One could imagine that with costs coming down al1 the time and with more automated methodologies being developed, that molecular characterisation of microbes may become routine in future. However, microbiologists with the relevant systematics skills, including molecular techniques, are in very short supply, and a recent review of microbial systematics ~ a i n i n g and research issues in the United Kingdom noted that the source of recruits necessary to succeed the present staff in culture collections alone was not readily identifiable (Review of UK Microbial Culture Collections, 1994). This projected shortfall of microbial biosystematists does not auger well for the pharmaceutical industry, In addition, it is important to have microbiologists with both modern taxonomic skills .plus field experience to more efficiently prospect for novel pharmaceuticals from microorganisms.

However, despite their sophistication, molecular methods do not indicate how many individuals of a species should be represented in screening collections. For example, through screening many different fungi over the years, it is apparent that there are variations in secondary metabolite production between different isolates of the same species from different substrates and in different habitats. It is not clear, however, whether these differences mirror the diversity of genotypes within a sampling site, or if the diversity is a result of habitat variability and epigenetic variability. Studies we have undertaken to examine this (e.g. Talbot et al., 1996) have highlighted the complex relationship that exists between genotype, environment, and phenotype as expressed in secondary metabolic products. It is thought that differences in macro- and micro-climatic factors influencing the fungal populations, differences in substrate patchiness, environmental disturbance, and limitations in the flow of fungal propagules within a habitat might allow more diverse populations of fungi to evolve in some habitats. Such examples highlight the benefit of exanîining more than one population of a species and of having ecological, in addition to systematic, knowledge of the organisms one is examining in order to maximise the chances of discovering new chemicals.

Other organisms

The interest of companies in screening new types of organisms has created additional biosystematic demands in these areas, particularly where many new or unidentifiable species are collected during routine work. Many countries have amateur taxonomists such as lepidopterists, ornithologists, botanists and so on who may be valuable as parataxoriomists (see below) for in situ work. However, outside of developed countries, and where many of the unusual organisms are being collected, there is often a dearth of professional taxonomic expertise available. For example, in my owri experience with fungi, there are few biosystematic mycologists in Australia, yet there are large numbers of fungi endemic to the country. The taxonomy of some groups of fungi may be relatively well covered due to their importance as plant pathogens, but the sheer magnitude of fungal diversity in conîparison to plant diversity (Hawksworth, 1991) means that any biosystematic fungril work required by AMRAD will almost certainly have to be done in conjunction with overseas organisations such as the International Mycological Institute (IMI).


The expansion iiito screening new types of organisms and the reorganisation of existing sample collections within pharmaceutical companies has created a need for these companies to use outside supply sources and biosystematic expertise, which may be in the form of biosystematists working within culture collections, museums, governnîent research institutes, tertiary institutions and specialised companies. This has coiîîe at a time when many of these institutes and companies are suffering governnîeiit funding cutbacks and are required to raise more of their own funding through conîmercialising their resources and expertise. It may be opportune that these changes are occurring at the same time, but it has also meant that several companies may be sharing the use of a limited number of suppliers andlor biosystematists. In some instances these suppliers and consultants must carefully cornpartmentalise their work to avoid potential confidentiality difficulties.

Screening at Source

Conducting initial drug screeriing at source can have a number of advantages. In underdeveloped countries the development of the countries' scientific infra- structures can be effected. In a recent plant supply agreement between the Australian pharmaceutical Company AMRAD Corporation Ltd. and the Government of Sarawak, AMRAD will provide training for Sarawak scientists to assist in the developnîent of a Sarawak-based natural products venture in collaboratioii with AMRAD. Other advantages over conventional ex situ screening operations include the ability to examine specimens while their volatile compounds are still present, and the ability to screen samples during different seasons and under different environmental conditions. Temporal and locational changes in chemical activity, and changes in the production of active compounds with stage of the life cycle are of importance in optimising bioprospecting strategies. Despite the fact that organisnîs rrirely occur in monoculture in nature, the search for unilateral effects has meant that little work has been aimed at detecting the production of bioactive conîpounds in mixed organism situations, which may include the effects of conîpetition, etc.

In situ or even relatively close to source screening offers the opportunity to examine novel metabolite production in more natural settings. For example, Slattery et al. (1996) have reported site-specific and ontogenic differences in the concentrations of bioactive conîpounds produced by tropical soft corals. Production of these conîpoiinds was directly correlated with predation levels, with the compounds uiidergoing enzynîatic conversion from inactive pre-cursors following grazing. However, to do at source screening effectively, there must be on-site ecological siiid biosysteiîîritic expertise available.

Parataxonomists

The training and use of parataxonomists is another approach to on-site chemical prospecting (Joyce, 1991). Parataxonomists are local people who are trained to beconîe field collectors and who are paid for their work. This approach has been used in the agreement between Merck & Co. and Costa Rica's Institut0 National de Biodiversidad (INBio) under which Merck has the right to new microbial, insect and plant drugs that may be found in Costa Rica's nature preserves in return for INBio having ri share of the royalties from any drugs developed (Reid et al., 1993).


Collections and Related Organisms

When bioprospecting for natural products from macroorganisms, large re- collections niight necessitate surveys of the distribution and abundance of organisms, as well as determination of the variation of drug content in different organisni parts and the fluctuation of content with the season of harvesting (see above).

If probleiiis are ericountered due to scarcity of the wild organism or an inability to adapt it to cultivation, a search for alternative sources may be necessary. Other species of the sanie genus or closely related genera can be analysed for drug content. For exaniple, during routine screening of plants within their collection for novel natural products, researchers at the Royal Botanic Gardens Kew discovered an anti-AIDS compound from the seeds of an Australian bean tree, Ca.stano.spermurn uustrule, a species with no close relatives in Australia. They also discovered that a South Anierican tree with similar fruit and flower, Alexa leiopetalu, rilso contriined the same novel compounds. It is thought that the trees probably shared a cotnn-ioii ancestor millions of years ago when South America and Austriilnsia were still linked (Vines, 1992). Instances such as these denionstrate the recliiirement for access to good database information when searching for nat~iral products.

With regard to niicroorgrinisnis, bioprospectors do not face the potential large scale recollection problenis that nlight be encountered with macroorganisms. However, they are fiiced with a paucity of information available concerning the taxonomic relationships between niicroorganisms and also information concerning their geographic distributions, For exaniple, Samuels & Rossman (1992) have claimed that despite the diversity of habitats within the tropics there are few fungi that are strictly tropic~il ;is coinprired to those that extend into subtropical and temperate zones. They poirited out that most genera of fungi are cosmopolitan as compared to plants, whiçh have liriiited distribu-tions, and that most fungal species are in fact only known froni single collections, thus making it difficult to assess their distributions. Where rinlimorph and teleomorph connections can be made, some teleomorphs are known froni one or a few tropical sites while their anamorphs are known to be cosniopolitan.

Culture Collections and Taxonomic Databases

A reçent review of n~icrobial systematics training and research issues (Review of UK Microbial Culture Collections, 1994) noted that industrial users of culture collections prefer a single serirchable electronic database format as used by the Anierican Type Culture Collection. In addition, the development of taxonomic databases in an electronic form for global access, is of vital importance to the continued search for novel natural products of pharmaceutical importance. This is especially so if it allows bioprospecting to be done on a world-wide basis by a global community of researchers, institutes and companies. It would be useful if these taxonomie datribases were of a multimedia type and held phenotypic, cheniotaxonomic and molecular data so they were of value to anyone requiring data of the aforenientioned types.


Summary

The increased interest in prospecting for new natural products of phannaceutical importance and the advent of extract libraries has created opportunities for screening many new types of organisms other than those that have been traditionally screened. At a time when a number of larger pharmaceutical companies are beginning to question the size and composition of their natural product saniple collections and even to stop natural product screening in some instances, a market has been created for smaller companies focusing on specific types of organisms. Thus, the ongoing requirement within the pharmaceutical industry for biosystematists continues.

There is a requirement for taxonomists with skills in areas such as modern moleculür techniclcies. These skills are required for reorganising the many tens of thousands of isolates housed in large Company collections into more meanin ful P and usefully sized collections. In addition, both within the larger and sma ler companies actively bioprospecting for new organisms, biosystematists with relevant ecological experience are recluired to enable more efficient bio-prospecting. Ecological experience is of importance in selection of organisms, whether macro or micro, but is of fundamental importance when attempting to isolate "unusual" microorganisnis or those from relütively unstudied habitats.

l n situ or close to source screening offers the opportunity to examine meta-bolite production in more natural settings. However, this requires the availability of on- site ecological and biosystematic expertise, which may be in the form of trained parataxonomists, especially in countries where there are few professional taxononiists.

Access to good electronic taxonomiç databases, in multimedia format and allowing globril riccess, is of vit:il in-iportance to the continued search for novel natural products of pharmaceutical value. This is especially so if bioprospecting is to be conducted on a world-wide büsis and by a global community of researchers, institutes and companies.

Looking to the future, more undergraduate and graduate training in taxonomy at tertiüry institutions is required to ensure that there are future generations of taxononiists to succeed todüy's taxonomists.

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May, R. M. (1992) How inany spccics inhabil the Earlh? Scientific American 267, 42-48. Reid. W.V., Laird, S.A., Mcycr, C.A., Gamez, R., Sittcnfcld, A., Janzen, D.H., Gollin, M.A.

& Juina, C. (1993) Biodiversify Prospecting: Using genelic resources for sustainable developrnent. Washington, D.C.: World Rcsourccs Instilulc.

Review of UK Microbiul Culture Colleclions (1994) London:HMSO. Samucls, G.J. & A.Y. Rossinan (1992) Microliingi in divcrse tropical habitals. In Absfracts of

the British Mycologicul Sociefy Tropical Mycology Symposium, University of Liverpool, 6-9 April 1092.

Slaucry, M., M.T. Haininan, I.A. Khan, T. Pcrry, D. Comfort Jr., W. Walkcr, J. Starmcr & V.J. Paul (1996) Chcinical variation in lropical sofl corals. In Absfracts of 37th Annual Meering of lhe Americun Sociefy of Pharmacogno.sy, Univcrsily of California, Santa CCW, 27-31 July, 1996.

Systcmalics Agcnda 2000 (1994) Systemufics Agenda 2000: Churting the Biosphere. Technical Reporl. Ncw York: Sylcrnatics Agenda 2000.

Talbot, N.J., P. Vinccnl & H.G. Wildman (1996) The influence of genoiype and environment on lhc physiologieal and melabolic diversity of Fusarium compactum. Fungal Genefics & Biology 22, 254-287.

Vincs, G . (1002) Crcalivc chcinisuy. Kew (Summcr), 22-25. Wildinan, H.G. (1991) Lilhiuin chloride as a sclcclivc inhibitor of Ï'richoderma spccies on

soil isolalion plarcs. Mycological Reseurch YS, 1364-1368. Williams S.T., M. Goodl'cllow, G. Aldcrson, E.M.H. Wcllinglon, P.H.A. Sncalh & M.J. Sacklin

(1983) Nuincrical classil'ica~ion of Slrepfomyces and relalcd gencra. Journal of Cenerul Microbiology 129, 1743- 18 13.


The BioNET-INTERNATIONAL Approac h Tecwyn Jones

Technical Secretariat, BioNET-INTERNATIONAL, Bakeham Lane, Egham, Surrey TW20 9TY, UK

Introduction

It matters not whether it is called taxonomy, systematics or biosystematics, the fact is that this branch of science that is dedicated to discovering, identifying, naming and classifying organisms and elucidating their relationships is fundamental to Our attempts to understand biodiversity, and is the biggest single demand of this pursui t.

What began at the Stockholm Conference on Man and the Environment in 1972 as a mildly keener appetite for biosystematics was transformed at Rio in 1992 into an insatiable hunger, just as the sçientific sustenance to satisfy it began to become scarce. Today it is doubtful if there are any "ologists" or "ists" within the realms of zoology and botany who do not have some biosystematic demands. Identifications in particular are urgently needed in ever-increasing numbers worldwide by so- called "natural sçientists" of every hue and inclination - not least those in the biodiversity-rich but resource-poor countries of the developing world.

In these nations whose biodiversity, whilst fragile, is seen to be vital to the world's well-being and constitutes the Earth's richest genetic resource, there is little, and in some cases no, biosystematic capability. This is largely a consequençe of history and of recent fin:incial policies of developed countries.

From early colonial days, and especially from the turn of the century, the biosystematic needs of developing countries have been met and satisfied, until very recently, by free biosystematic services provided by the major world centres of expertise. These services, with particular emphasis on providing authoritative identifications and related advice, continued uninterrupted as colonialism fell away and these countries gained their independence. Whilst these services lasted there was no cause or motivation for developing countries to invest their scarce high- level manpower and firiancial resources in developing expensive biosystematic capabilities. I t would have been economic folly, perhaps, in view of competing demands on national budgets, to attempt to create indigenous capabilities in this very speçialised field whilst they were available free from elsewhere.

Sadly this comfortable arrangement was not to last. In 1993-94 as a result of worldwide recession, and the advent of new financial policies which required developed country institutions to become income-earning and self-supporting, the free services of the expert centres were increasingly withdrawn. They were replaced by a system of graduated charges for identifications and for al1 other services rendered, which, whilst favouring developing country clients, and being modest


relative to the actual costs of providing them, proved to be be-yond the means of developing country customers.

As a result, developing countries found themselves to varying degrees devoid of biosystematic services at the very time when they most needed them, i.e. when they, as adherents to Agenda 21 of Rio, and as signatories to the Convention on Biological Diversity, were attempting to meet their inter-national obligations. They were faced with a biosystematic crisis, and there was a biosystematic impediment to the pursuit of national programmes for sustainable agricultural development, conservation and wise use of the environment and biodiversity.

This was not unexpected. It had been foreseen by al1 concerned in the late 1980s when donor assistance had been sought in vain, to subsidise the services needed. It was indeed out of growing concern for a solution to the forthcoming çrisis that in 1991, an answer, which proved acceptable to donors, expert centres and developing countries alike, was devised in the form of BioNET-INTERNATIONAL. This initiative, which was launched through CAB INTERNATIONAL in June 1993, is a strategy for enabling developing countries to establish and sustain realistic self- reliance in biosystematics, and to do so in the technically best and most cost- effective way.

BioNET-INTERNATIONAL is concerned with helping developing countries to acquire and maintain the scientific skills, the collections of organisms and their related knowledge, and the technologies needed to provide the vital biosystematic support for national progrrimmes for conservation and sus-tainable use of their environment and biodiversity and sustainable agricultural development. BioNET- INTERNATIONAL is also a facilitating mechanism for the broader interventions needed to assist developing countries to achieve full implementation of the Convention on Biological Diversity.

Structure

BioNET-INTERNATIONAL is comprised of a senes of inter-linked sub-regional networks (LOOPs) of developing country institutions, supported by a consortium of developed country expert institutions known as BIOCON, and managed by the BioNET-INTERNATIONAL Consultritive Group (BICG) and its Coordinating Commi ttee (BICC) and its Technical Secretariat (TECSEC).

LOOPs (Locully Orgunised und Operuted Partnerships)

LOOPS are at the very core of BioNET-INTERNATIONAL. They are Technical Cooperation Networks (TCNs) dedicated, through SOUTH - SOUTH cooperation, to mobilising, pooling and optimising the use of existing biosystematic skills and

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National Tnstitutes represented on the TCN

by their NACI - Lines of communication

Figure 2. I-lypotlieticril six member LOOP or Techiiical Cooperrition Network (TCN)

BIOCON (The Cotzsortium for Teclirzical Support for BioNLT- INTERNATIONAL)

This coiisoitiiiiii of the world's migor ceiitres of biosysteiiiatic expertise and resources is designed to piovide the iiiforriiation, skills, riisterinls aiid techiiologies needed by developiiig couiitry subregions to ricliieve serilistic self-relinnce in biosystematics. I t is the soiirce of techiiical support for doiior-fiinded programmes for capacity biiildiiig iiiid hiirnriii resource developinent iii the BioNET- INTERNATIONAL LOOPs of the developiiig world, i.e. NORTH - SOUTH cooyerntioii.

This consortitini is beiiig cieated, piece-nieal tliroughout tlie world as developed country iiisti tiitioiis begiii to collriborate to make their diverse resources üvailüble. The first siibsegioiinl coi~sortiuiii EuroLOOP, with some 75 institutions spread throiighout 25 coiintries, was established in 1994 niid is iiow expaiiding as it enibai-ks on the tusk of iiiventoryiiig the sesources i t lias to offer to developiiig country LOO13s. A secoiid BIOCON initiative hris begiiii i n South Africa (SafriLOOP) wliere tlie Agricultiirril Keserircli Council niid other bodies are collriborating. A thiid BIOCON LOOP is conteinplated iii the Australasirin region to serve tlie needs of tlie Pacifie LOOP of BioNET-INTERNATIONAL. A resource fii 1 Noslli Aiiieric;iii LOOP is i i i prospect.


Work Progrunt~~tes of LOOPS

The subrcgional LOOPs have four priorily work programmes viz:-

Estublishntent undlor Etzl.latzcement of Information and Communication Services

There is a nccd 10 updalc and expand hard copy library resources at designated centres of exccllcncc (CES) of LOOPs, Tor example wilh major reference works, taxonoinic monographs and relevant serial journals, and information technology nceds to bc providcd 10 thcsc CES for intcr-centre nelworking and linkages with the NEC1 and TECSEC. Databases and database access arrangemenls with major world cenlrcs nccd LO bc cstablishcd and Inlernct acccss, including E-mail facilities, are rcquircd.

An cîficicnt inl'ormiiiion scrvice providing al1 relevant new knowledge is needed covcring traditional laxonomy, molccular tcchniques, ncw rccords, current pest dislribulion inaps and A l iind A2 quarantine pcst lists, and incidcnce and threats of exolic pcst iniroducrions. Rclcvant inl'orma~ion on natural cnemics/biological control and biorcincdialion agcnls nccds to bc acccsscd and kcpt up to datc.

Training o~Bio,s~~.scer~tuci.st.s und Technicians

This, ll-ic mosl subsianlial pi-ogrammc of thc LOOPs for the Sorcsccable Suture, will involvc:

Updriling and upgrading o l cxisling cxpcrtise through appropriale training of the present subregional specialists at overseas universities and institutions andlor at local subrcgional ncadcinic and scicntil'ic cénlrcs. Also supplcmcnlary training is needed in specialised taxonomic areas, for example pests, natural enemies, post- harvest pest identification and diagnoses, quarantine organisms.

Training of tcchnical support staff in prcparatory tcchniques and curatorial pracliccs, collcciion managcmcnt, dalabasc managcment and information retrieval syslcms, illuslraiivc tcchniqucs and devclopmcnt of elcctronic products. Elcclronic Lcacliing cousscs and training matcrial nced to bc provided to the CES.

Reluhilitation ofCo1lectiorz.s und Establishment of New Resources

Thcsc major programincs addrcss ihc inadcquacics and necds of cxisting preserved and living rcfcrcncc collcclions and îacililics, including buildings, slorage units, working aincnilics, sccui-ily, curalorial tcchniques and the suslainability of the physical condilion OS collcciions and lhcir allcndanl records. Thcse programmes also addrcss lhc Laxonoinic nccds ol' collcclions 10 oplimisc Lheir valuc as working resoui'ccs l'or lhc LOOP.

Development and Applicution of New Technologies

Thcsc pi'ogramnics aiin 10 makc cxisling uscr-fricndly taxonomic tools, for example, clcctronic aids 10 idcnlific:ilions, and compcndia available to thc LOOP specialists, and to cn:iblc LOOPs to commission and/or dcvclop products Lhat arc tailor-made for


their own rcquircmcnts. More elcctronic and paper-based identification aids need to be commissioncd and ncw tailor-made products need to be devcloped jointly by overseas spccialists and subrcgional biosystcmatists for their own requiremcnts.

Progress

LOOPs have been andfor are being established in the following sequence:-

1. Caribbcan LOOP - CARINET (22 countries) - Dcccmbcr 1993 2. European BIOCON- EuroLOOP (25 countries) - June 1994 3. Southem Africa LOOP - SAFRINET (12 countries) - Scptcmbcr 1995 4. South Pacillc LOOP- PACINET (26 countries) - February 1996 5. South East Asia LOOP - ASEANET (9 countries) - August 1996 6. Wcst Arrica LOOP - WAFRINET (18 countries) - Scptcmber 1996 7. East Africa LOOP - EAFRINET (6 countncs) - October 1996

By the end of 1997 BioNET-INTERNATIONAL L O O P S will have been established o r activated by governments in the Caribbean, Africa, Asia, South Pacifiç and Latin America enibracing:

Amcrican Samoa Angola Argcntina Bahamas Bangladesh Barbados Bclizc Bcnin Bhutnn Bolswana Brunci- Darussalam Burkina Faso Cabo Verdc Cam bodia Camcroun Chilc China Sicrra Lconc Singaporc Solomon Isla~ids Sri Lanka Surinam Swaziland

Colombia Cook Islands Costa Rica C6tc d'Ivoirc Dominica Doininican Rcpublic Erilrca Ethiopia Fcdci-atcd Statcs of

Microncsia Fiji Frcnch Polyncsia Gambia Glinria Guam Guinca-Conakry GuinrSc-Bissau Tanzania Tchad Thailand Togo Tokclau Tonga

Guyana Haiti Honduras India Indonesia Jamaica Kenya Kiribati Laos Lccward Islands Lesotho Malawi Malaysia Maldivcs Mali Marshall Islands Mauretanic Trinidad & Tobago Tuvalu Uganda Vanuatu Vcnezucla Vielnam

Mauritius Mozambique M yanmar Namibia Nauru Ncpal New Caledonia Niger Nigeria Niue Pakistan Palau Papua New Guinea Philippines Pitcairn RSA Senégal Wallis et Futuna Wcstem Samoa Windward Islands Zam bi a Zanzibar Zimbabwe

A small but vcry supporlivc group of donors including the United Nations Devclopmcnt Programme (UNDP) and the bilateral agencies of Holland, Denmark, Gcrmany, Swcdcn, Swiizcrland and thc Uniicd Kingdom and the intcrgovemmental


agcncies of thc Commonwcrillh Sccrctariat, and CAB INTERNATIONAL have enablcd the Fcasibilily Sludics and LOOP Formulalion Workshops ncedcd to establish subrcgional LOOPs. In Lhis way Lhesc LOOPs have bccn crcalcd with full and forma1 support by ~ h c govcrnmcnls conccrncd, 10 whom thcy bclong. They are thus accordcd soinc priorily in lhc nalional dcvclopmcnt plans of Lhose who own them. Donor funds arc now fonhcoming from such agcncies as lhc Swiss Dcvelopment Coopcralion Agcncy (SDC) and the Overscas Dcvelopmenl Adminislration (ODA) of the Unitcd Kingdom and from Dculschc Gescllschaft für Technische Zusammenarbcil (GTZ) in Germany and from the Europcan Union's Ccntre for Tcchnical and Rural Coopcralion (CTA) 10 cnable the implcmcntation of the work prograinmcs of cstablishcd LOOPs. In addition a conlribulion of SFr 2.9 million by the Swiss Dcvclopmci~~ Coopcralion agcncy io the BioNET-INTERNATIONAL FUND has cnriblcd lhc ini1i;ilion of a BioNET-INTERNATIONAL Fellowship Schcmc, ail Informa~ion Supporl Scrvice and has assislcd lhe operations of the Tcchnical Sccrclarial and Nclwork Coordinating Inslitulcs.

The rcsponsc of thc international donor communily to furthcr substanlial funding proposals suggcsl lhiit lhc wholc global nctwork could bc in place wilhin the next threc ycars, and BioNET-INTERNATIONAL'S dcvelopment phase may be complclcd by 2006. By thn l limc lhcrc should be cstablishcd wilhin the subregions of the dcvcloping world a substaniial biosystemalic resourcc 10 support national prograrnmcs.

BioNET-INTERNATIONAL is a mcchanism bascd firstly on subregional self-help i.c. Soulh - Soulh Coopcration, 10 mobilisc and pool and oplimisc the use of existing rcsources and sccondly, on Norih - South Coopcration 10 ~ransfcr knowledge, skills and Lcçhnologics 10 dcvcloping subrcgions. Thc BioNET-INTERNATIONAL concept also ci~visrigcs ~ h c provision of csscntial biosystcmatic scrviccs 10 dcvcloping counlrics during Lhcir lransilion 10 sclf-rcliance, wilh donor support bcing provided 10 the LOOPs as inlcgral parts of nalional dcvclopmcnl prograrnmcs.

Key Issues

The achicvcmcnt of BioNET-INTERNATIONAL'S objcclives within Lhe 10-year time horizon cnvisagcd can only bc conlemplalcd bccause of, and lhrough, the use of new tcchnologics - no1 lcasl clcclronic aids 10 identi-fications. Thcse require first and forcmost lhc availability of kcys writlcn by world cxpcrts for lhc groups of organisms conccrncd, which can thcn bc convcrlcd into cleclronic forms suilable for a whole spcclrum of uscrs - from lhc up-slrcam rcsearchcrs/biosystcmalists, to more downstrcam praclilioncrs suçh as applicd biologisls, plant prolcclionists. quarantine personnel, ctc.

Thesc lcchnologics makc biosyslcmalics morc available to, and more useable by, a much Iargcr coininunily of scicnlisls and Lcchnicians, and enablc thesc to become compctcnl in idcntifying lhc organisms of rclcvance to Lhcm. Expcrience has shown thal as such kcys bccoinc availablc thcir impacl leads to a demand for olhers, and as thcsc kcys uliiiniilcly dcpcnd on thc skills of ~ h c Lradilional biosystcmalist, the dcmand for Lhcsc cxpcrls is also incrcasing. The nccd for biosyslcmatisls has ncver bccn grcalcr and Lhcir rolc in improving ihc wclfarc of human-kind has nevcr bcen as wcll pcrccivcd as il is today.


The European Network on Systematic Biology

Walter Los* and Steven Blackmore** *Zoological Museum Amsterdam, P.O. Box 94766, 1090 GT Amsterdam, The Netherlands;

"The Natural History Museum, Cromwell Road SW7 5BD London, UK

The Europcan Nclwork on Systematic Biology was established in 1994 by the European Sciencc foundation (ESF). It is a typical consequence of the policy of the ESF to promole challcnging scienlific developments. As a body whose members come from thc mrjjor rescarch councils and academies of 20 European countries, the ESF aims at Lhc advance of European cooperation in basic research and to advise on research and scicnce policics. The ESF-council does this by identifying fields of interest that nccd special attention, and bringing together a group of scicntists in a nelwork. Such a nclwork is cxpccted to bring together the key-scientists, and to define a rcscarch programmc. The budgct to support ESF networks has a restricted lifctimc, bu1 should providc a basis for funher activities

Systcinalic biology is prcsently characlerized by various new and exciting devclopments. Thcsc dcvclopmcnls have an impact on science and Society. Biological diversity is widely rccogniscd as a vital rcsource that will play a crucial role in enabling countrics 10 move towards a morc sustainable path of development. Many thousands of species, both ond land and in the seas, provide food, shelter, clothing, medicincs, commerce and othcr cssenlial scrvices. Species are also thought to play an as yct poorly dcïincd rolc in an array of ecological processes which support life on earth, such as thc cycling ol' carbon, nitrogcn and water. Systematic biology provides the l'und;imcnlül framcwork for al1 biological study, and is concerned with discovcring and dcsciibing biological diversity, elucidating evolutionary relationships (phylo-gcny) bctwecn organisms, and construcling hierarchical classifications that rcflcct thcse rclationships. New Lheories, melhods and othcr innovations have rapidly changcd thc scienlific disiplinc of systcmalic biology.

The Europcan and widcr systcmatic biology community is at a crucial point in its history, as i t confronls lhc vas1 scientific challenge posed by the need to understand and conscrve the Earth's rapidly declining biological diversity. By establishing the ESF nctwork, il was acknowlcdgcd that the challenge can be met only by international collaboration which maximiscs thc bcnefils of excisting expertise and systematic resourccs. I L was Surthcr rccognized Lhat Lhe initiative Systematics Agenda 2000: Charting the Biosphere (1994) is an important refcrence for our European activities. Europe has a special responsibility with respect to the attention for systemalic biology. This scicnlilic ficld bcgan in Europe and, wilh more than half of thc world's biological collcclions, European systematic inslitutions are a global scicntific and widcr cullural rcsourcc. Howevcr, the communily of biological syslemalists is no1 currenlly organised in such a way as to mect the scientific challenges poscd. This silualion is an outcome of the traditionally seperate disciplines (bolany, cntoinology, zoology, terrestrial versus marine biology) and the widely docu-mcntcd dccline in support 10 systcmatics relative to other fields of science. An


effective communication and collaboration between the still significant group of Europcan scicntists should be promoted.

The Network and Its Objectives

The ESF Nctwork on Systcmatic Biology is not a restricted group of persons, but a loosely organizcd nclwork of scientists that meet in workshops on some defined topics. Thcsc workshops are advertized widely, and are open for every interested scientist. A coordination committee takes care of al1 organisational matters, and will promote the further dcvclopment of new initiatives that are expected to emerge from the workshops. This committee defined some objectives, which were endorsed by the ESF Council. Thc objcctivcs are the following:

To unite European systematists through food communication and to create a dccentraliscd systcin:itic rcsource. The network wants 10 direct itself a1 the level of individuril scicntisls, lcading to institutional collaboration and coordination. Establishing lasting lincs of communication will enable the decentralised systcmalic collcclions of Europe to function effcctivcly as a single scientific facilily. To promotc thc ~ransfcr of information. Computerized taxonomic systems are of vital impoizancc, no1 only as a new tool to manage and organise the vast amounts of information and expcrtisc, but also as a service to a wider audience of user's. Coordination and communication between many different initiatives are conditional to accclcratc progrcss. To cxplorc thc rclationship bclwccn morphological and molecular approaches to taxonomy. Molccular mcthods arc yiclding new insights into different systematic relations. Too oftcn howcver, the proponents of the two - morphological or molcculnr - ripproachcs tcnd to work separately, with different goals. The disciplinary boundarics should bc broken down cffcctivcly. To considci. ncw dcvclopmcnts in systematic theory and practice. Systematic analysis has becn fast moving ficld in recent years. Various new methodologies and Lcchniqucs pur a prcssurc on cxcisting theorics, and are attractive for new gcnerations of scicntisls. Thcre is a necd to find solutions for the problem to keep abrcast of' thcsc dcvclopmcnts, and to broaden the application of systematic analyscs 10 ncw arcas of biology. A possible approach is by defining a European rcscarch agcndri, which inay bc subjcct for additional financial support. To cstablish priorilics for and considcr ways of promoting the training of systcmatisw. Many rcccnt rcvicws have highlighted the concem about the lack of provision of training for systcinatisLs now that the subjcct is not widely taught at universities. the age stnicture of the systematic community is weighted towards oldcr individunls and Lhcrc is concem that not enough younger scientists are bcing rccruitcd into thc discipline. At the same lime, an important role of Europcün systcmaLists is ~ h c transfcr of taxonomic expertise. The network should consider solulions rathcr than to run courses.

The rcsulls OS the considerations of the nctwork's activities may have various foms. Intitiativcs of coopcr:itivc inslitutcs or individual scicntisls will be encouraged, but it is also cxpcclcd to producc policy papcrs on systematic priorilies. They may aid dccision-makcrs in national and European research institutions and funding agencies, including thc ESF and thc Europcan Commission. The coordination committce of the

Biology International NO35 (August 1997)

Nclwork promolcs lhc cxchangc of information and lhc dcbate about the state of affairs via thc workshops, a ncwslcllcr, and a Wcbsitc (hoslcd by Thc Nalural History Muscum, London; Inlcmcl addrcss: hllp://www.nhm.ac.u/csf/.

Workshops

Wiih support from Lhc ESF, a scrics of workshops was planncd. A first exploratory workshop was organiscd in 1994 (France) on the subjccl of Molecular Taxonomy. This small workshop pu1 the basis of a much larger workshop "Molccules and Morphology in Syslcmalics" in Paris (March 1997). In 1995 the îirsl large workshop was organizcd in Lcidcn, Thc Nclhcrlands, in collaboralion with the Rijkshcrbarium Leidcn, lhc Linncan Socicly of London and thc Systematics Associalion. The overall objcclivc was 10 idcnlilj, what Europe's spccial rcsourccs in systematic biology could conlribulc 10 documcnling and undcrslanding biological diversity. The kcy elcments of lhis goal arc cxprcsscd by thc inilialive Syslcmalics Agcnda 2000. An important rcsull ol' lhis workshop was Lhe broad parlicipalion from different disciplines, bringing Logclhcr vicws froin riIl arcas of systcmatics. The participants agreed to focus on morc coopcraLivc cl'lor~s Srom lhc diffcrcnt disciplincs. The Leidcn workshop sct the tone for various new developments that emerged from that time. In March 1996 the coordinaling commillc organiscd a workshop on "Disscminaling Biodiversity Information" in Ainslcrdam, Logclhcr wilh lhc Zoological Muscum Amsterdam and the Expcit Ccnlrc for Taxonomic Idcntificalion. Since the topic of taxonomic inforinalion proccssing wils idcnlificd as a key area, the workshop provided an exccllcnl plalforin 10 announcc thc lalcst dcvclopmcnts and 10 discuss the opporlunilics for coininuiiicnlion and coopcration lowards ncw Europcan initiatives building upon currcnl work.

Thc lasi workshop in Scplcmbcr 1997 (Crclc) will focus on new directions in Syslcmalics, and is cxpccicd 10 dclinc ü Europcan rescarch agenda, to bc submitted 10 thc ESF and olhcr funding agcncics. ForlunaLely, new rcscarch projccts and other devclopmcnls do no1 wnil l'or Lhis final workshop. The mcrc fact that scientists from diflcrcnt disciplincs mcl cach olhcr al Lhe previous workshops, resultcd in new conlacis and ncw idcas 10 coopcralc. Thc coordinating committee does not keep stalislics on Europcan coopcralion syslcma1ics, but various scicnlists observcd a considcrablc progrcss in bringing thc syslcmalic communily togethcr.

Organizational lmprovements

Togcthcr wilh thcsc dcvclopincnts, lhc Nc~work is also conccrncd about a stronger organizalion of thc ins1ilulion;il bodies and organizalions. IL has becn considcrd to slriil a Europcan Associalion of Syslcmalics. This may havc an inlcrcsting fulure, but no1 unlil ~ h c kind and lhc numbcr of pan-Europcan activilics will nced such an organizalion. II' il is cxpcclcd LhiiL disci-plinary Europcan associalions want to find a common plalform, and i f thc individual syslemalists are willing 10 support such a plalfoim, il inny the ~i inc 10 takc aclion. Another inslilutional iniliative was in time. Aficr a suggcslion from thc coordinaling commiltcc, the ESF supported a plan to eslablish a Consorliuin of European Taxonomic Facililies. This consortium should bring logclhcr lhc lnrgcr scicncc-bascd taxonomic inslilutions, wilh the objective to promolc acccss 10 ils collcclions. IL is cxpcctcd ~hrit thc proccdure to cstablish this consorlium wilh inLcrcslcd inslilutions will gct ils shapc during 1997.


Gaetano SALVATORE (1932- 1997)

1 first met Gaetano (Nino) Salvatore in Bethesda, Md., some fifteen years ago. We were both living i n Building 20 on the Campus of the National Institutes of Health (NIH), where both of lis were spending a Fogarty Scholarship-in-Residence. 1 was immediately impressed by his warmth, his intelligence and his humor. Our common Neapolitan background made Our mutual understanding extremely rapid. During the time we spent together at NIH we met frequently, Nino and his wife and closest collaborator Marisa often exchanging very enjoyable visits with my wife and myself. At that time, Fogarty scholars had a weekly seminar and 1 still remember well a seminar of Nino on thyroid hormones as an example of clarity and scholarship.

A few years later, when Nino became President of the Stazione Zoologica of Naples, he invited rile to join the Scientific Council. This gave us the opportunity to meet again on a regiilar basis. The meetings of the Scientific Council were not only interesting, but they were also very pleasant. On those occasions, the "Musici dell'Accluario" gave beautiful concerts i n the stunning room with the frescos by Hans von Müreés. This initiritive of Nino revived the original cultural role of the Stazione, a glorious Institiition that Anton Dohrn founded in 1872 in order to prove that Darwin was right.

1 hrid already been a Member of the Scientific Council of the Stazione many years before, when Alberto Monroy was the Director. At that time, everybody thought that the problems that had accumulated over the years by the Stazione had no solution. When Nino took over, the situation of the Stazione Zoologica was almost desperrite. I t took the indomitable energy of Nino to achieve the miracle of resurrecting the phoenix from its ashes. Nobody else could have achieved this tour de force. But "impossible" was a word missing in Nino's vocabulary.

When Nino became the President of the Stazione, he had already behind him a very bright career. Hüving obtained a medical degree at the very early age of 23, he became a full Professor of General Pathology when he was only 31. His major scientific interest was endocrinology and, more specifically, the thyroid hormones. He had been successfiilly working on this subject for a long time, at one important point in his cltreer with Jean Roche at the College de France in Paris.


But Nino's personality had many facets. Indeed, he played an important role at the University of Naples, where he was the Dean of the Faculty of Medicine and Surgery for twelve years, and also the Director of the Center of Endocrinology and Experimental Oncology. At the National level, he also was the driving force behind the revision of the study course i n Medicine in Italy, as well as the Chairman of the Committee of Biotechnology and Molecular Biology of the National Research Council of Italy. At the International level, Nino was the Chairman of the Italian Committee of the International Union of Biological Sciences. He fostered international cooperütion in the biomedical sciences, and organized with Howard Schachnian a Conference on this subject at NIH. I t is especially sad thnt he will not be with us at the 4th International Conference of Marine Biotechnology, which he organized and which will take place in Southerii Italy in September 1997.

Nino received many recognitions for his activities. He was a Member of the Accademia dei Lincei and of the Finiiish Acadenly of Medicine, a "Chevalier dans l'Ordre NationLi1 du Merite" of France, a Fogrirty-Scholar-in-Residence of NIH and a recipient of the Feltrinelli Prize, to quote only a few of these honors.

Whrit is incredible is that during the past fifteen years Nino carried out al1 of his activities in spite of ri serious heart condition, which had required surgical intervention. He never let his health interfere with his work. In fact, he continued his activities unti l the lrist driy, which he spent in Rome at a Senate hearing, dying of a heart attack shortly after he reached his home in Naples in the evening.

The loss of Nino is a very hard blow to Italian research, but he provided an example which will stimulate others to continue his work. His many friends from al1 the parts of the world will always remember him for his warm humanity, his volcanic energy, his Neapolitan inirigination and his generosity, which had no limits. He really wûs what in Nriples is crilled "un signore".

Giorgio Bernardi Institut Jacques Monod, CNRS, Paris, France


IUBS Financial Statement for the Year 1996

l STATEMENT 1: Balance shcel at December 31, 1996 (Expressed in US$)

ASSETS: cash and Ilanks

CrCdit Lyonnais, Paris in USS in FF in USS (Dcposii. Accounl)

Merrill Lynch, New York(in US$) In US$

Profil on porifolio Other Receivables

LESS: LI ABILI'I'IES Sundry Creditors

EXCESS OF ASSEI'S OVER LIAI%ILIrI'IES Represeiited by

Gcncral Sund Mandaiory Rcscrvc

STATEMENT 2: Income and Expenditure for the year ended December 31, 1996 (Expresscd in US$)

1. INCOME ICSU Subvcnlions Special Subvcn~ions froin Ini.crriai.iona1 Organisalions Contributions froin Nalionri1 Mci'i~bcrs Salcs of publicalions Intercsls and Dividcnds Profit on porlfolio Orher Incoinc Total Inconie

2. EXPENDITURK Business Meetings General Asscinbly, Excculivc Coinrnillce & Officers Mcclings Publications Scientifle Prograninies and Activities Scientilic Meetings; Syinposia ColIoquia Granls lo IUBS Scicntific Prograinincs Rcprcsentations al scicntific mcctings Contribulion lo othcr organisations Scientific Activilics of IUBS Sçicntific Mcrnbcrs Secretariat Salaries &Rclalcd chargcs General OSfîcc cxpcnscs Office equiprncnl and supplics Bank chargcs & Loss ovcr Exchangc Audit (Intcrnal audit and lcgal Cccs Total Expenditure

Excess ot' Inconie over Experiditure 50 109 1


BIODIVERSITY AND SAVANNA ECOSYSTEM PROCESSES A Global Perspective

Edited by O. T. Solbrig, E. Mcdina and J. F. Silva. Publishcd by Springer, Ecological Studies No 121, 1996 (233 pagcs).

This book addrcsscs the role of species diversity in thc function of savanna ecosystems. It shows that savannas are enonnously diverse and that four factors determine the function of savanna ecosystems: plant available moisture, p lan t avai lablc nutricnts, f i re and herbivores.

CONSERVING BIODIVERSITY FOR SUSTAINABLE DEVELOPMENT

Edited by P.S. Rainakrishnan, A. K. Das & K. G. Saxena Published by INSA, Delhi, India, 1997 (246 pagcs).

This book consists of thc procecdings of two IUBS sponsorcd symposia convcncd by P. S. Ramakrishnan in 1995 on: (1) Biodiversity- Gcnes to Ecosystcms: Towards Suslainablc Management" held in Delhi, India,, and (2)"Managing Bio- diversity for Sustainablc Dcvclopincnt" organized during FASAS Congress in Kuala Lumpur, Malaysia.

FISH COMMUNITIES IN LAKE TANGANYIKA

Edited by H. Kawanabc, M. Hori & M. Nagoshi. Published by Kyolo University Press, 1997 (298 pagcs).

This book providcs a comprchcnsive and up-to-date rcvicw of ihc major findings of the Japancsc-Aîrican rcsearch Lcam which has bccn studying tlic biology of the fishes in thc rocky litloral zonc of Lake Tanganyika sincc 1979. Il. covers a wide variety of biological perspectives including fish bchaviour, ecology, gcnetics and evolulion, and providcs a unique insight in10 Lhc intcraclions within a tropical frcshwatcr community.

As such, it represents a useful resource for ecologists, conservationists and managers.

ISLANDS Biological Diversity and Ecosystem Function

Edited by P. M. Vitousek, L. L. Loope and H. Adsersen. Published by Springer, Ecological Studies No 1 15, 1995 (238 pages).

This volume deals with the components of biological diversity on islands and their patterns of variation; the modern threats to the maintenance of biological diversity on islands; the consequences of island biology and ils modification by humanity rcgarding aspects of ecosystem funclion; the global implications of islands for conservation and how they can hclp one to understand the processes inducing changes throughout the world.

FUNCTIONAL ROLES OF BIODIVERSITY A Global Perspective

Edited by H. A. Mooney, J. H. Cushman, E. Mcdina, O. Sala, E.-D. Schulze. Published by Wilcy SCOPE Scries No 55, 1996 (493 pages).

This volume contains the final synthesis of the SCOPE/DIVERSITAS programme dealing with the ecosystem functioning of biodiversity addressing two basic aucstions: fi) Does biodiversity "count" in system processcs, e.g. nutrient retention, decomposition, production, including atmos- pheric feedback and in face of global change; (2) How is systcm stability and resistance affectcd by species diversity, and how wil l g loba l change af fec t these r e l a t i o n s h i p s ?

BIOLOGY INTERNATIONAL€¦ · Biology International No 35 (August, 1997) Tumors in Lungfish The...

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