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Fungus Populations M Marine Waters and Coastal Sands of the Hawaiian, Line, and Phoenix Islands' CAROL WRIGHT STEE LE2

Fungus Populations M Marine Waters and Coastal Sands of the Hawaiian, Line, and Phoenix Islands' CAROL WRIGHT STEE LE2

Populations m Marine Waters and Coastal Sands of the Hawaiian, Line, and Phoenix Islands' CAROL WRIGHT STEE LE2

ABSTRACT: Saprophytic and facultative parasitic fungi present in the coastal waters and adjacent pelagic areas of the Hawaiian Islands, and in coastal sands of the H awaiian, Line, and Phoenix islands, were isolated by plating methods. Isolates from all areas sampled indicate that abundant and varied fungus popu­ lations do exist in these environments. The number of fungi obtained from the inshore was seven that obtained from the oceanic zone. The fungus Anreobasidium pltllttla11S ( De Bary) Arnaud was isolated repeatedly from oceanic waters. A comparison is made between the genera and the average number of isolates per liter of water known from the Atlantic with those found in this study of the Pacific Ocean. The number of fungi isolated from sand samples of the different islands ranged from 2 to 1,600 per gram. diversity was evident throughout the samples. The leeward Hawaiian islands had a higher aver­ age number of isolates per gram than any other island group. In conclusion the problems of defining a marine fungus are discussed.

OCEA NIC AREAS in different parts of the world also are very limited. These include a few rec­ have been shown to be habitats for marine ords of higher fungi collected in the Marshall fungi (Johnson and Sparrow, 1961) . Investi­ Islands (Rogers, 1947) , the Society Islands gators, however, have usually concentrated on (, 1957, 1958) , and Raroia in the Tua­ particular groups of fungi by use of selective motu Archipelago (Cooke, 1961); Phyco­ isolation methods ( Barghoorn and Linder, mycetes recovered by plating soils of the 1944; Moore and Meyers, 1959; Jones, 1962) . of Bikini, Eniwetok, Rongerik, and Ronggelap Only one extensive analysis of marine waters (Sparrow, 1948); and Ascomycetes and Fungi for a general fungus population is known, and Imperfecti from dung and soil samples col­ it was made in the northwestern subtropical lected by Olive in the Society Islands (Peter­ Atlantic Ocean (Roth et al., 1964) . References sen, 1960) . Consequently, as Cooke points out, to the occurrence of fungi in the Pacific Ocean the geographic distribution of fungi occurring are found (1 ) as incidental to studies of bac­ on the islands of the Pacific is poorly known. teria in marine water (ZoBell, 1946) ; (2) in Although studies have been initiated on the studies of specialized fungi such as lignicolous soils of the Hawaiian Islands ( Baker, 1964) fungi (Cribb and Cribb, 1955 , 1956, 1960; and the (Marsh, 1965) , no study Kohlmeyer, 1960; Meyers and Reynolds, 1960) has been made of fung i occurring in marine and those on (Cribb and Cribb, 1955 , waters and intertid al environments of these 1956, 1960); and (3) in studies of particular islands or elsewhere in the central Pacific. This kinds of fungi , e.g., in Japanese investigation was undertaken to determine the waters (Kobayashi, 1953) and path ogenic spe­ occurrence and distribution of the saprophytic cies (Van Uden and Castelo Branco, 1961 ) . and facultative parasitic fungi which constitute Reports of fungi from terrestrial environments the fungal populations of these habitats. of islands in the central and southern Pacific

1 Prepared from a thesis submitted in partial ful ­ MATERIALS AND METHODS fillment of requireme nts for the Master of Science degree at the University of H awaii. Collection 2 Department of , San D iego State College, San Di ego, Californ ia, 92115. Manuscript received Isolations for fungi were made from 59 June 20, 1966. water samples and 50 sand samples collected 317 318 PACIFIC SCIENCE, Vol. XXI, July 1967

from inshore areas of the Hawaiian Islands and the millipore filter method (Roth et al., and adjacent pelagic environments. Seventeen 1964) . sand samples were taken from the Phoenix and Standard materials and procedures were used Line islands. The water samples were taken in the pour-plate method. Amounts plated for from four zones: the surf or spray zone; the the water samples ranged from 0.5 ml to 5.0 inshore neritic zone, 2-200 m from shore; the ml. Dilutions of 1: 10 to 1: 100,000 were used offshore neritic zone, 300 m to 2 km from for sand samples. Some samples were plated shore; and the oceanic zone, 2 km or more off upon return to the laboratory; because of the shore. Of these water samples 56 were taken distance between most sampling sites and the at the surface and 3 were taken at depths laboratory, however, most of the plating was down to 600 m. The sand samples were done 24 to 48 hours after collection. All sam­ taken from a depth of 2 inches in the supra­ ples were kept under refrigeration until plated. tidal, intertidal, and subtidal zones as delimited A control plate, uninoculated, was set up for by Hedgpeth ( 1957) . The sample sites were each medium for every plating. Plates were selected to include a variety of shore environ­ also exposed to the air during plating opera­ ments: leeward, windward, or areas of special tions to determine the level of air contamina­ interest (e.g., South Point, Hawaii, the south­ tion in the laboratory. ernmost point in the Hawaiian Islands; W ai­ Materials for the millipore filter method kiki Beach, probably the most frequently used included sterile -ester membranes with beach on Oahu; and Midw ay Island, the north­ 0.45 !! porosity (Millipore Filter Corp., Bed­ ernmost inhabited island of the Hawaiian island ford, Massachusetts) . Th e samples were run chain.) Other samples were obtained as oppor­ through the millipore filter apparatus using a tunity offered : those from Kure Island, the vacuum pump. The membranes with retained Phoenix Islands, the Line Islands, and the fungal elements were cultured on selective me­ open ocean. Figure 1 gives the geographical dia in presterilized pastic petri dishes. The location of the water and sand collection sites. amount put through the filter ranged from The surface water samples were collected in 100 ml to 600 ml per sample. Controls were sterile 600-ml glass bottles with plastic screw included for testing sterility and air con­ caps. The closed bottles were immersed in the tamination. water, then opened and allowed to fill with Several kinds of media were used: sodium water, closed underwater and brought to the caseinate agar (BBL 01-549, Fred and Waks­ surface. The depth samples were taken by send­ man, 1928, modified by Potter, 1957), Roth's . ing a plastic water sampler of the van Dorn isolation medium (Roth et al., 1964), Fell's ( 1956) type to the designated depths. After agar (Fell et al., 1960) and mycobiotic the sampler was brought to the deck of the agar ( DIFCO 0689-02) . All media except the ship, a sterile 600-ml bottle was filled with mycobiotic agar were made with water col­ water from the sampler. lected at the sample sites. Bacterial growth was All sand samples were collected with sterile controlled by the incorporation of 0.05% chlor­ implements and placed in sterile 100-ml jars amphenicol (Chloromycetin, Steri-Vial N o. 65, with plastic screw caps or in sterile poly­ Parke, Davis and Co.) . Dilutions were made ethylene bags (NASCO Whirl-Pak, Hydro Prod­ with McIlvaine's buffer solution, pH 7.0 ucts Co., San Diego, California) . (Machlis and Torrey, 1956). The mycobiotic Salinity measurements were obtained by use agar was made with 250 ml sea water and of Quantabs S051 (Linayer Corp., Detroit, 750 ml of distilled water in to retain Michigan) . Temperature was determined by the selectivity of this medium in which chloro­ standard centigrade thermometer, and depth mycetin is already incorporated. by standard oceanographic methods. The plates were placed in paper bags, sealed with adhesive tape, and incubated at 20°_ Isolation 24°C. Pour-plates were incubated for three Two principle means of isolation were em­ weeks and the millipore filter plates for 10 ployed: the pour-plate method (Salle, 1954) days at room temperature (200-24°C). from Central Pacific-STEELE 319

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FIG. 1. General geographical location of marine water and coastal sand collection sites. X , Marin e water collection sites; 0 , coastal sand collection sites. 320 PACIFIC SCIENCE, Vol. XXI, July 1967

Oth er methods of isolation, such as baiting successfully were Czapek-Dox agar (DIFCO (Johnson and Sparrow, 1961) , spread-plate 0339-01) and "V-S" juice agar (Wickerham technique (Bu ck and Cleverdon, 1960) and et aI., 1946). cellulose plates made by placing a piece of sterile filter paper over an before RESULTS inoculating (Baker, 1964) were attempted on some samples. The pour-plate and millipore Populations in W ater filter methods, however, proved superior for Variati ons in salinity and temperature for this study. all water sample sites was slight, ranging be­ tween 30 %0 and 35 %0 for salinity; between Identification 20 0 and 25DC for temperature. Inasmuch as Some fungi were identified directly from sampling was done over the period from June the isolation plates by a technique employing 1964 to May 1965 this temperature range also pressure sensitive tape (clear tape, reflects the slight variation characteristic of a Scotch N o. SOO, Minnes ota Mining and Manu­ tropical . facturing Co.). This meth od is described by All sample sites yielded fungus colonies Roth, Orpurt, and Ahearn (1964) . Other (Table 1); 44 of the samples had counts of fungi were transferred to various selective me­ 50 colonies or under, whereas only 15 gave dia to encourage sporulation. Those used most counts over 50. The standard plate count was

TABLE 1

ZONAL D ISTRIBUTION OF FUNGI IN M ARINE WATERS AND COASTAL SANDS

AVE RAGE AVERAGE NUMBERNUMBER NUMBER NUMBER OF ISOLATES OF ISOLATES WATER SAMPLES SAMPLES PER ML SAND SAMPLES SAMPLES PER GM M ain H awaiian islands Main Hawaii an islands Kauai Kauai 2 .16 5 218 Oahu O ahu Surf zone 9 .10 Suprat idal zone 3 185 Inshore neritic zone 9 .83 Intertidal zone 3 6 In shore bay zone 3 .28 Tidal pool zone 2 72 O ffsho re neritic zone 3 .61 Subtidal zone 3 14 Polluted zone 3 3.18 Maui Mau i Intert idal zone 36 Surf zone .17 H awaii Hawaii In tertidal zone 6 77 Surf zone 3 .09 Leeward H awaiian isl ands Inshore neritic zone 5 .43 Kure Leeward H awa iian islands Intertidal zone 8 41 M idway Midway Su rf zone 1 .20 Sup ra tidal zone 1 1000 In shore neritic zone 4 .031 Intertidal zone 6 790 Oc eanic, Johnston Island 9 .066 Other leeward O ceanic, Oahu 7 .029 H awaiian islands Intertidal zone 12 1220 Surf zone total 16 .12 Line Isl ands Inshore neritic zone total 18 .34 Int ertidal zone 4 150 Inshore bay zone tota l 3 .28 Ph oeni x Islands Offshore neritic zone tota l 3 .61 Intertidal zone 13 80 Oceanic zone total 16 .04 5 Polluted zone total 3 3.18

GRAND TOTAL 59 .14 TOTAL 67 Marine Fungi from Central Pacific-STEELE 321 not significant for the diluti ons used. There­ number of isolates than either the surf or fore, the number of isolates from the total oceanic zones. A very low average number of number of milliliters used in plating is re­ isolates was obtained from both oceanic regions. corded as an average isolation return per ml The offshore oceanic area near Johnston Island of water samples. The average number of iso­ returne d more isolates than the comparable zone lates from anyone sample ranged from 0.06 near Oahu , but both areas had about the same to 3.94 isolates per ml, with the average for number of species. A ureobnsidinm pullnlans samples at 0.14. The number of species ranged and Rbodotorula spp. were common to both from 1 to 17 per sample. More than 50% of oceanic sites. These fungi were among those the sites, however, returned only 2 to 7 dif ­ predominant in all isolations from water feren t species (Steele, 1965) . The predominant (Table 2) . genera and species by percentage of occur­ As might be expected, samples from the rence are listed in Table 2. polluted areas off Oahu had the highest aver­ Table 3 lists the 126 species of fungi repre­ age number of fungi : 3.18 per ml. This was senting 59 genera which were isolated from an area of diverse speciation. Members of the the water samples plated. The percentage of were common, as were species occurrence represents the number of water sam­ of A spergillus, , and Cepbalo­ ples in which a particular fungus occurred in sporism. reference to the total number of samples ana­ lyzed. A tabulation of species isolated from Populations in Sand the six zones sampled shows that they can be From 67 sand samples plated from four dif­ ranked in descending order for number of feren t zones, 134 species of fungi representi ng species per zone as follows: inshore neritic, 70; 71 genera were recovered (Table 3). The fre­ surf, 56; polluted zone, 28; oceanic, off John ­ quency of predominant isolates by species is ston Island, 23; oceanic, off Oahu, 20; and given as percentage of occurrence (Table 2) . offshore neritic, 13. The inshore neritic zone The average number of isolates per gm, ob­ was the richest area, having a higher average tained by the standard dilution plate counting

TABLE 2

PREDOMINANT G EN ERA AND SPECIES IN WATER AND SAMPLES

PERCENTAGE * PERCENTAGE OF OF WATER OCCURRENCE SAND OCCURRENCE

Yeasts 4 5.8 A spergillus u/entii 50 .7 Rh odotorula spp. 27. 1 spp . 44 .7 Fusarium spp . 22 .0 spp. 25 .3 Cephalosporium curtip es 22 .0 Penicillium spp . 22 .3 cladosporioid es 16 .9 niger 20 .8 C. epiphyl/um 16 .9 20 .8 Helminsbosporium anomalum 16.9 M egasler sp. 17.6 T richoderma lignorum 15 .2 M asoniel/a grisea 1604 A spergillus niger 13.5 A spergillus spp. 14.9 A. wentii 13.5 A . terreus 13.4 pullulans 13.5 A. ustus 11.9 spp . 11.8 lignorum 11.9 A spergillus spp. 11 .8 Cladosporium cladosporioide s 11.9 Black yeasts 11 .8 C. epipbyllum 11.9 Pestalotia spp. 10.1 Cephalosporium roseo-griseum 11.9 Cladosporium berbarum 10 .1 C. spp . 11.9 A spergillus versicolor 10 .1 C. ID A Penicillium spp. 10.1 C. curtipes ID A Penicillium liIacinu m ID A • The percentage of occurrence represents the number of water or sand samples in which a particular fungus occurred in reference to the total of 59 water samples or 67 sand samples analyzed (Orp urt, 1964) . 322 PACIFIC SCIENCE, Vol. XXI , July 1967

TABLE 3

GENERA AND SPECIES ISOLATED FROM MARINE WATERS AND COASTAL SANDS

PE RCEN TAGE PE RCEN TAGE OCCURRENCE OCCUR RENCE IN WATER IN SAN D NAME SAMPLES SAM PLES

PHYC OMYCETE S echin ulate Thaxter 1.4 C. sp. 1.4 globosus Fischer 1.4 Rbizopus nigrlcans Ehrenberg 1.6 2.9 Syncepbalastrum racemos um (Cohn) Schroeter 1.6 1.4 ASCOMY CET ES ChaeJomium oliuaceum Cooke and Ellis 1.4 C. sp. 1.6 M elanomma sp. 1.4 M elenospore lagenaria (Pers.) Fuckel 1.4 M . sp. 1.4 Microescus intermedius Emmons and Dodge 1.4 M. trigonosp orus Emmons and Dodge 1.4 sp. 5.0 2.9 Sp orormia sp. 2.9 BASIDIOMYCET ES Sp. indet. 1.6 DEUTEROMYCETES Sphaerop sidales Amerosporium sp. 1.4 sp. 5.0* Conioth yrium fuckelii Sacco 1.6 Cytosporlna sp. 3.3 sp. 1.4 D iplodina sp. 1.6 M acrophoma sp. 1.6 sp. 3.3 Phoma bibernica Grimes 3.3 2.9 Phoma spp . 11.8 7.1t Pbomopsis sp. 1.6 Pbyllosticta sp. 1.6 Piggotia sp. 1.6 Pyrenocbaeta sp. 1.6 1.4t Spha eronaema spinell« Kalchb . 1.6 Sp oronemn sp. 1.4 Melanconiales Pestalotia sp. 10.1 5.9t Pbylactaena sp. 1.6 Mon iliales Sporobolomycetaceae sp. 2.9:1: Mon iliaceae Acremoniu m sp. 1.4+ Acrostalagmu s cinn abarlnus Corda 1.6 1.4 Aleurisma cam is (Brooks and Hansford) Bisby 1.4t A llescb eriella crocea (M ont.) H ughes 1.6 Aspergillus amstelodami (Mangin) Th orn 1.4+ Marine Fungi fro m Central Pacific-STEEL E 323

TABLE 3 (continued)

PERCE NTAGE PERCENTAGE OCCU RREN CE OCCU RREN CE INWATE R IN SAN D NAME SAMPL ES SAMPLES A. caespitosus Raper and Th orn 1.4 A . candidus Link 404::: A. carneus (van Tiegh) Bloch. 404 ::: A. clava/us Desm. 2.9::: A . elfusus Tiraboschi 1.4::: A . {lavipes (B ainier and Sartory) Thorn and Chu rch 3.3 2.9 A. f/avus Link 1.6* 104 A. fumigatus Fres. 1.6 404 ::: A. granulosus Raper and Th orn 7.1 A . itaconicus Kinosh ita 1.6 * A . janus Raper and Thorn 1.6 404 A . lucbuensis Inui 1.6 2.9 A . micro-uirido-citrinus Cost. and Lucet 1.6 A . nidulans (Eidam) Wint. 1.4 A . niger van Tiegh . 13.5* 20.8::: A . niueus Bloch. 104 A . ocbraceus W ilhelm 3.3 A. oryzae (Ahlburg ) Cohn 1.6 2.9 A. panamensis Raper and Thorn 2.9 A. prolijerans G.Smith 1.6* A. restrictus G. Smith 1.4 A. tu ber (Spieckermann and Bremer) Th orn and Chur ch 1.4 A . sulpbureus (Fres.) Th orn and Church 3.3 1.4 A. sydou/i (Bain. and Sart.) Thorn and Church 3.3* 7.1::: A. tamarii Kita 1.6* 2.9+ A. terreus Th orn 3.3 1304::: A. unguis (Emile-W eil and Gaudin) Th orn and Raper 1.4::: A . UJtUJ (Bainier) Thorn and Chu rch 1.6 11.9::: A . versicolor (Vuill. ) Ti raboschi 10.1* 5.9::: A. urenzii Wehmer 13.5* 50.7t::: A . spp . 11.8* 14.9 Botryopbialopbora marina Linder 2.9 Cephalosporium acremonlum Corda 3.3* lOAt ::: C. asperum Marchal 1.4 C. coremioides Raillo 1.4 C. curtipes Sacco 22.0 * l OA C. bumicola Oud emans 3.3* 1.4 C. roseo-griseum Saksena 6.7* 11.9+ C. sp. 1.6 11.9 viride Grove 1.6 1.4 fimbl-iatum Gilman and Abbott 1.4 Malbranchea sp. 1.4 M oeszia sp. 1.6 M onilia brunnea Gilman and Abbott 1.4 M onoci//ium sp. 1.6 5.9::: [uslsporus Saksena 1.6 * P. uarioti Bainier 1.6 2.9 Penicillium albidum Sopp 1.4 P. breui -campacturn D ierckx 3.3 P. canescens Sopp 3.3 P. caseicolum Bainier 1.6 1.4 P. cbarlesii Smith 1.6 1.4::: ------_. 324 PACIFIC SCIENCE, Vol. XXI, July 1967

TABLE 3 (continued)

P ERCENTAGE PERCENTAGE OCCURRENCE OCCU RREN CE IN WATER IN SAND NAME SAM P LES SAM PLES P. cbermesinum Biourge 1.4 P. citrinum Thorn 1.6 8.9:1: P. commune Thorn 1.6 P. corylopbilum Di erckx 1.4:1: P. cyaneo-iuloum Biourge 1.6 P. cyaneum (B ainier and Sartory) Biourge 1.6 P. janthinelJum Biourge 3.3 P. kapuJCinskii Zaleski 1.4:1: P. lanosum Westling 6.7 1.4 P. lanoso-coeruleum Thorn 5.0 P. lilacinum Thorn 6.7 * 10.4 P. miczynskii Zaleski 1.4 P. nigricans (Bainier) Thorn 8.4 8.9+ P. notatum Westling 5.0 P. oxalicum Currie and Thorn 1.6 1.4 P. piscarium Westling 1.4 P. pu rpu rrescens Sopp 1.6 P. raciborskii Zaleski 1.6 P. rotundum Rape r and Fennell 1.4:1: P. simp licissimum (Oud.) Thorn 1.6* P. steckii Zaleski 1.6 4.4:1: P. oelutinum van Beyrna 2.9 P. spp . 10.1* 22.3t:l: Rbinotricbum sp. 1.4t::: Scopu lariopsis breuicaulis Bainier 4.4:1: S. brumpti] Salvanet-Duval 1.4:1: S. carbonaria Morton and G . Smith 1.6 S. croci van Beyrna 1.4:1: S. fimicola (Cost. and Mat.) Yuill. 1.6 2.9 S. sp. 1.6 7.1 Sepedonium sp. 1.6 2.9 Spiearia sim plieissima Oudernans 1.6* Sporotrichum epigaeum Brunard 2.9 T richoderma album Preuss 3.3 T. glaucum Abbott 1.6 2.9 T . koningi Oudernans 5.0* 1.4 T . llgnorum (Tode) Harz 15.2* 11.9 Trinacrium sp. 1.4 Tritiracbium album Lirnber 1.4 T. purpureum (Saito) Beyrna 3.3* Vericosporium sp. 1.4 Ve rticillium candelabrum Bonorden 1.6 V. terrestre (Link) Lindau 3.3 1.4 D ernatiaceae Acrostapbylus sp. 1.4 Acrotbeca sp. 1.4 [asciculata Cooke and Ellis 1.6 1.4 A. geophila D aszewska 1.6 A . bumicola Oudemans 3.3 A. tennis Nees 3.3 Aureobasidium mansonii (Ca st.) Cooke 1.4:1: A . pullulans ( De Bary) Arn aud 13.5* 8.9t:l: A . sp. 1.6 4.4:1: Mar ine Fungi from Central Pacific-STEELE 325

TABLE 3 ( conlinued)

PERCE NTAGE PERCEN TAGE OCCUR REN CE OCCU RRENCE IN WATER IN SAND NAME SAMPLES SAMPLES sp. 1.4 Carenu laria sp. 1.6 Cbalaropsis sp. 1.6 Cbloridium sp. 1.6 Cladosporium clados porioides (Fres.) de Vries 16.9* 11.9t:l: C. epipoyllum Persoon 16.9* 11.9 C. berbarum (Persoon) Link 10.1 8.9t:l: C. lignicolum Corda 3.3 C. spp. 10.4 Cordana sp. 2.9t Curuularia geniculata (Tracy and Earle) Boedij n 6.7 1.4:1: C. lnt erseminata (Berkeley and Ravenel) Gilman 3.3 C. pnllescens Boedijn 3.3 4.4 C. subu lata (Nees) Boedijn 3.3* C. tetramera (McKinney) Boedijn 3.3 D endrypbion sp. 1.6 Gliobotrys albooiridis von Hohnel 1.4 conuoluta (Harz) Mason 8.9 Gonytricbum macrocladum (Sacc.) Hughes 1.6 G. sp. 1.4 H ansjordia togoensis Hughes 1.6 H. sp. 1.6 H elmimb osporium anomalum Gilman and Abbott 16.9 1.4 H . satloum Pammel, Kin g and Bakke 1.6 H eterosporium sp. 1.4 H ormodend rum cladosporioides (Fresenius) Sacco 1.6 H umicola grise« Tragen 1.6 H . lanuginosa (Griff and Maubi.) Bunce 1.4 H. nigrescens Omvik 1.4 H. sp. 2.9 M acfO sporium cladosporioides Desm. 1.6 M. sacrinaejorme Cavara 1.6 Masoniella grisea (Smith) Smith 5.0 16.4:1: M egasler sp. 17.6tt M enispor« apicalis Berk . and Cur t. 1.6 spbaerica (Sacc.) Mason 3.3 8.9t Oidiodendron citrinum 1.4 O. gri seum Robak . 1.4 sp. 1.4 byssoides Persoon 1.4 P. blspidula (Pers. ex Pers .) Mason and M. B. Ellis 1.4 Phialop hora sp. 8.4 25.3t :l: Scolecotricbum sp. 1.4t Stacbybotrys atra Corda 1.4 S. lobulata Berkeley 4.4 Stempbylium botryosom W allr ath 1.6 S. macrosporoideum (Berkeley and Broome) Sacco 3.3 T orula allii (Harz) Sacc. 1.6 T. lucifuga Oudemans 4.4t T. sp. 1.4:1: T richocladium sp. 1.4 Z ygosporium masonli H ughes 4.4 Stilbaceae 326 PACIFIC SCIENCE, Vol. XXI, July 1967

TABLE 3 (continu ed)

PERCENTAGE PERCENTAGE OCCURRENCE OCCURRENCE IN WATER IN SAND NAME SAMPLES SAMPLES D idym ostilb e sp. 1.6 Grapbium sp. 1.4 H arpograpbium sp. 1.6 Synnematium [onesii Speare 3.3 1.4 Tuberculariaceae didymum (Hartung) Wollenweber 2.9t C. radicicola Wollenweber 1.4 pu rpurascens Ehrenb . 1.6 E. sp. 2.9 Corda 1.6 F. spp. 22.0* 40tt Hym enella sp. 4.4t Myr otheeium roridum Tode 3.3 M. oerrucaria (Alb . and Schw.) Ditmar ex Fr. 1.4 M. sp. 1.6 1.4 Yeasts Rbodotorula spp. 27.1* 1.4 Orange yeasts 1.6 8.9tt Pink yeasts 2.9t Black and orange yeasts 1.4 Black yeasts 11.8* 8.9tt Yeasts 45.8* 20.8tt Mycelia sterilia (Dematiaceae) 28.8* 37.3t t Mycelia sterilia (Moniliaceae) 16.9* 40.ltt • Isolated from oceanic zone. t Isolated from the Line Islan ds. :I: Isolated from the Phoenix Islands . technique, is given in Table 1. A total of 37 frequently in water and sand are listed. Among samples had fewer than 100 isolates per sam­ these, 9 are common to both areas although of ple; the remaining 30 samples had more than different rank for percentage of occurrence; 100 isolates per sample. The sand of the lee­ 7 occur in water and not sand , and 8 occur in ward Hawaiian islands had the highest number sand, not in water. Of those common to both of isolates, over 500 per sample, but the nurn­ water and sand, yeasts, aspergilli, and peni­ ber of different species was lower than in com­ cillia were common to all sand samples. Neither parable samples from the Phoenix Islands . Rh odotorula spp. which is penultimate in rank The sands returned from 1 to 35 species per for water, nor Aureobasidizan pI/lilt/aIlS, also sample. The majority yielded from 3 to 9 frequent in water samples, was predominant species each. The intertidal sands of the main for sand samples. Hawaiian islands returned the highest number The control plates poured when both water of species, ·a total of 68. Black Sand Beach, and sand samples were plated showed no Hawaii , and Kaena Point, Oahu, each yielded growth. Only one was observed on a totals of 16 species. The supratidal zone of plate exposed to determine the level of air Kuhio Beach, Oahu had the highest number contamination in the laboratory. of species for sites in that zone: 35 species among 16 genera. The zone total was 53 spe­ DISCUSSION cies. The subtidal zone returned the lowest number of species, only 22. Isolates obtained from water samples indi­ In Table 2, the 18 fungi occurring most cate that abundant and varied fungus popula- Marine. Fungi from Central Pacific-STEELE 327

tions do exist in this environment, but that Sphaeropsidales, of the , were frequencies vary with zones. The oceanic zone frequently found in the Oahu samples. These had fewer isolates than did the other zones fungi are parasitic on . The polluted studied in the pelagic region. Thi s result was area contained much floating debris which expected, inasmuch as oceanic regions are could serve as a source of these fungi. known to have lower populations of marine Some water samples were taken from tidal than do regions closer to land. Of pools in the intertidal zone. In every case there the 59 water samples, 50 were taken from areas was a high number of isolates. Intertidal pool that are strongly influenced by the presence of populations may be affected by higher temper­ oceanic islands, whereas the other 9 were from atures, higher organic content, salinity levels, areas away from any shore. Areas near or wave action. One area, however, that of a islands are known to support abundant and large bay on the windward side of Oahu, did varied marine . Ships may also be a source not return a high yield of isolates. This area of water pollution, and therefore modified has a high fresh-water run-off which reduces populations might be expected in shipping the salinity at the surface as much as 5 %0dur­ lanes. ing the rainy season. Even though the area Differences can also be observed between had a lower popul ation than expected, it did the two oceanic locations studied, as reflected have great diversity, explained perh aps by the in differences in kind of fungi present but run-off factor. The high number of aspergilli not in numbers. The oceanic area off Johnston and penicillia isolated was to be expected Island contained more yeasts than did surface because of their proclivity for sporulation, samples obtained near the island of Oahu. cosmopolitan habitat, and their great adapt­ The latter had more fungi which would be ability. A good percentage of these might be classified as terrestrial, such as aspergilli and run-off and/or air contaminants introduced into penicillia. The high yeast population observed the water. from the oceanic areas is in agreement with The fact that more fungi were isolated from the findings of both ZoBell (1946) and of the sand than from the water supports the Fell and Van Uden ( 1963). Members of the well-known observation that microbial popu­ Rb odotorula were isolated consistently lations are higher in relation to fixed surfaces. from all water samples, including the depth Examination of Table 3 also shows that the samples. Roth et al. (1962) have noted the general population of sand is quite different common occurrence of these yeasts in oceanic than that of water. localities, an observation now confirmed by The high number of isolates from the sand these studies for the Pacific Ocean. samples taken from the leeward Hawaiian When the three zones-surf, inshore neritic, islands and the few species among them is in and offshore neritic-are compared, a correla­ direct contrast with the low number of isolates tion is found between numbers of isolates per and high number of species in the samples ml and location of zone. This is not unex­ from the Phoenix Islands and the Line Islands. pected. This correlation is particularly clear in There could be many reasons for this. The the data for these zones in Oahu. The surf elapsed before plating the leeward sam­ zone, which is an unstable area with constant ples was greater than for those of the Phoenix wave action, returned 0.10 isolates per ml com­ and Line islands. Variations in bird population, pared with 0.33 for offshore neritic and 0.83 temperature, and hum idity, and shore stability for inshore neritic zones. Species of Cnrunlaria, among the leeward Hawaiian islands, may be A lternaria, and H elmintbosporium were iso­ critical factors controlling fungus populations. lated repeatedly from the inshore neritic zone. If the bird population serves as a control, a This zone, in the area of Oahu, is richer in survey for keratinophilic and coprophilous spe­ number of species than is the same zone on cies might be rewarding. Such a survey should Hawaii. This might be due to pollution, as also extend to other islands with large bird Oahu has a greater population and has a major populations. Another factor influencing fungu s port for shipping. Members of the order populations, as reflected in the high number 328 PACIFIC SCIENCE, Vol. XXI, July 1967 of species returned from the Phoenix and Line Most of the Phycomycetes and the Ascomy­ islands, is the fact that Gardner Island, Wash­ cetes were isolated from sand. These fungi are ington Island, and Palmyra Island are, or have known to adhere to substrates. Onl y one species been, inhabited. predominates over the others, Asp ergillns The supratidal zone gave the highest average toentii, as shown in Table 2. When plating four num ber of isolates per gm. This zone is a very samples on mycobiotic agar from beaches that stable area, a mixture of soil and sand. When are local tourist attractions on Oahu, one po­ the average number of isolates is compared for tential was found : Micl'OasClts inter­ the supratidal, intertidal, and subtidal zones on medius, which has been isolated from a number Oahu, the intertidal displays the lowest average of soils by passage. Several species of number of isolates, which may be explained this genus are known to be etiologic agents of by the influence of constant washing by the dermatophytoses and in man waves. The tidal pool area of the sand, like (Barron et al., 1961 ). the tidal pool area of the water, is characterized It is interesting to compare the results ob­ by the presence of more fungi than are found tained by Roth et al. (1964) in the Atlantic in the surroundi ng areas of each zone. with the results obtained from this study in the Two of the sand areas sampled are unique Pacific. Roth took 227 water samples and iden­ among sands for their color and composition, tified 41 genera among his isolates. This study namely a green sand beach and a black sand encompassed 59 water samples and resulted in beach. The black sand beach had 259 isolates the identification of 59 genera. Of these, 29 distributed through 12 genera and 200 species, genera were common to both lists. The Atlantic while the green sand beach had 28 isolates, in list included 11 genera not reported in this 10 genera and 12 species. Although 4 genera study; this study includes 29 genera not repor ted were common to both, only 1 species, Asper­ by Roth et al. ( 1964) . gillm terreus, occurred at both sites. Both Table 4 shows the difference in average num­ beaches are on the island of Hawaii. One rea­ ber of isolates per liter between the samples son for this difference may be that the black taken in the Atlantic Ocean and those taken in sand beach is continually exposed to contamina­ the Pacific Ocean. tion from people and their litter, while the Furthermore, it is a striking fact that no Pa­ green sand beach is in a very remote location. cific sample, either water or sand, was without Another reason could be the difference in the fungi, whereas Roth et al. ( 1964 ) recovered chemical composition of the sands. Black sand fungi from only 80% of their 227 samples. is formed from basalt lava rock and cinders, Because they did not include sand samples in and green sand is formed by the release of oli­ their study, comparisons can be made only be­ vine crystals which are in the basalt lava rock. tween water samples. The maximum number of

TABLE 4

C OM PARISON O F N UMBERS OF ISOLAT ES FROM THE A TL ANTICO CEAN AN D THE PACI FIC O CEAN

ATL ANTIC OCEAN ( ROTH ET AL. 1964) PAC IFIC OCEAN ( 196 5)

NUMBER AVERAGE NUMBER NUMBER AVERAGE NUMBER O F ISOLAT ES OF ISOLAT ES DEPTH SAMPLES PER LITER DEP TH SAMPLES P ER LITER

500 to 5 11.1 600 m 13.0 1000 m 17 12.1 300 m 6.0 9 4.9 300 m 10.0 2 1.0 Surface 12 15.5 Surface 18.0 to 79 17.5 55.0 500 m 43 15.1 30.0 16 3.0 73.0 Marine Fungi from Central Pacific-STEELE 329 species per offshore sample in the Atlantic was certain isolate is a marine fungus. There is no 6. In the Pacific the number ranged from 1 to single diagnostic test. The criteria which have 11. Although the maximum number of species been suggested are summarized by Roth et al. per Pacific sample exceeds that reported for the (1964) and may be stated as: (1) the isolate Atlantic, the total number of species found in must grow and reproduce exclusively or pre­ each area is approximately the same: 133 species dominantly in the sea or on intertidal substrata, in the Atlantic compared with 127 in the Pa­ or (2) the isolate must grow and reproduce at cific. The diversity of genera is notably higher an optimal level in the normal salinity of the in the Pacific samples. Samples from both areas . None of these criteria can be supported yielded fungi that were not identified. without qualification. There is still no means of There are both differences and similarities concrete demonstration of growth and repro­ between the kinds of fungi obtained from the duction of a fungus in vivo in marine environ­ two regions. A total of 30 species were common ments except for those fungi growing on nat­ to Atlantic and Pacific waters. Neither Asco­ ural (and introduced) substrata. If growth and mycetes nor Basidiomycetes were reported for at normal salinity levels is ac­ the Atlantic. From the Pacific water 2 Ascomy­ cepted as a criterion, this allows for the inclu­ cetes and 1 Basidiomycete were recovered. Roth sion of fungi found in salt lakes (Anastasiou , et al noted the low incidence of Phycomycetes, 1963) . Moreover, Gray (1963) has shown that reporting only 6. Five species were identified in many fungi of terrestrial origin are capable of this Pacific study, but only 2 were from water better growth in sea water media than in fresh samples. Species of Sphaeropsidales occurred in water media. If growth is limited to natural similar numbers in both oceans: 7 in the At­ substrata, then the possibility of free-living ma­ lantic and 5 in the Pacific, but only 2 species rine fungi is excluded. were common to both. In comparing the species As more investigations of of fungi which are predominant in each area, are undertaken, the number of genera and spe­ some species are found on both lists. Roth et al. cies isolated from them will undoubtedly in­ distinguished 8 fungi each from the eulittoral crease. Many of the genera found in the Pacific and oceanic water samples as their dominant are known from other marine locations, e.g., species. Of these, was species of Aureobasidium, , Phoma, most common in the oceanic zone and it did not , Diplodia, Epicoccum, , occur in their eulittoral list. In the Pacific, this Cladosporium, Alternaria, and fungus was common in water but ranked sev­ (Johnson and Sparrow, 1961). Repeated isola­ enth among 18 species. Cladosporium species tion, however, is not confirmatory evidence, but (sic) were the most common fungi in the At­ is only suggestive. Roth et al. (1964) contend lantic eulittoral samples, and occupied second that, until further distributional and physiolog­ place in the oceanic list. Two species, Clado­ ical data are obtained, fungi so isolated should sporium cladosporioides and C. epiphyllum, be regarded as of incidental occurrence in the ranked high among those frequent in both wa­ sea. Nor is the rare isolation of a species from ter and sand samples of the Pacific. Tricho­ only marine habitats reliable evidence, as this derma lignorum, the last species on the Atlantic may reflect only the randomness of the sam­ list of eulittoral dominants, is absent from the pling and the samplers. Curiously, Dendry­ corresponding oceanic list. In the Pacific it oc­ phyiella armaria Nicot., which is dominant in curred in both water and sand samples, although Atlantic eulittoral samples and known only much more frequently in water than in sand. from marine sources, was not among the Pa­ In summary, the differences suggest that the cific isolates. Conversely, Botryophialophora Pacific fungus population is different from that marina, also known only from marine sources, of the Atlantic. was found in the Pacific but not in the Atlantic. Having established the fact that fung i can In conclusion, a working definition for a be isolated from Pacific as well as Atlantic marine fungus is proposed : A marine fungus water and shores, there still remains the prob­ is one which is capable of producing successive lem of how one determines whether or not a generations by sexual and/or asexual means in 330 PACIFIC SCIENCE, Vol. XXI, July 1967 natural oceanic waters or on intertidal substrata. from Biscayne Bay, Florida and adjacent ben­ Until experimental means of proof are devised, thic areas. Limnol. Oceanog. 5:366-371. the data presented here will serve as contribu­ --- and VAN UDEN, N. 1963. Yeasts in tory evidence for the distribution of fungi iso­ marine environments, pp. 329- 340. In : C. H. lated from marine habitats. Oppenheimer, ed., Symposium on Marine Mi­ crobiology. Charles C Thomas, Springfield, ACKNOWLEDGMENTS Illinois. FRED, E. B., and S. A. WAKSMAN. 1928. 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