Pacific Science (1988), vol. 42, nos . 1-2© 1988 by the University of Hawaii Press . All rights reserved

Evolutionary Relationships of the Hawaiian and North American Telmatogeton(Insecta; Diptera: Chironomidae)

LESTER J. NEWMAN!

ABSTRACT: Species of Telmatogeton and the closely related genus Para-clunio generally live on the rocky shores of the intertidal zone. Species of Tel-matogeton have evolved from the marine environment into torrential freshwaterstreams of the Hawaiian Islands . An analysis of the banding sequences of thepolytene chromosomes of species of Telmatogeton and Paraclunio from theHawaiian Islands and North America suggests that there were at least twoseparate invasions from the marine to freshwater environments. One is throughthe marine species T. japonicus to an undescribed freshwater species found oneast Maui . The other invasion requires a marine hypothetical species that gaverise to the Hawaiian freshwater species T. abnormis and T. torrenticola.

SPECIES OF THE FAMILY Chironomidae aregenerally found in aquatic or semiaquaticenvironments. Species in only 13 genera ofchironomids, including the Telmatogetonand Paraclunio, occupy the marine environ-ment (Hashimoto 1976, Sublette and Wirth1980). The life cycle of most species of Tel-matogeton and Paraclunio is spent on therocky shores of the intertidal zone. In theHawaiian Islands , species of Telmatogetonhave evolved from the marine environmentinto torrential freshwater streams (Wirth1947).

Wirth (1947, 1959) describes the discoveryand naming of species of the Telmatogetonand Paraclunio . The genus Telmatogeton wasestablished by J. R. Schiner in 1866, and thefirst species, T. sancti-pauli, was describedby him in 1868 from specimens collected onSt. Paul Island in the Indian Ocean . D. W.Coquillett described the second species,T. alaskensis, from specimens collected fromYakutat, Alaska, during the Harriman Ex-pedition of 1899. The first Hawaiian fresh-water species was described by F. W. Terryin 1913. J. J. Kieffer established the marinegenus Paraclunio in 1911 for P. trilobatus,collected on the coast of California, and in1915, J. R. Malloch moved T. alaskensis tothe genus Paraclunio. The major morpholog-

1 Portland State University, Department of Biology,P.O. Box 751, Portland, Oregon 97207.

56

ical feature distinguishing the genera is asexual dimorphism of the front femur andtibia . These structures are narrow and undif-ferentiated in males and females of Telmato-geton and in females of Paraclunio. The frontfemur of Paraclunio males is broad, and anangular projection of the distal portion ofthe femur fits into a corresponding notch inthe base of the tibia. Because of the morpho-logical similarity of the two genera, the genusParaclunio should be synonymized with theTelmatogeton (Peter Cranston, personalcommunication). The Telmatogeton and Para-clunio are thus essentially treated together inthis discussion.

The natural history and ecology of theTelmatogeton and Paraclunio are describedby Kronberg (1986), Morley and Ring(1972), and Robles (1982). Two importantpoints relative to the current discussion arefeatures that lead to the isolation of popula-tions and the adaptation of species to asevere environment. Adults of both marineand freshwater species exhibit a peculiarswarming behavior; they are generally seenrunning about on intertidal rocks and onstream banks seeking mates. Adults are notstrong fliers and thus may have limitedvagility. Adults of the marine species are be-lieved to be short-lived ; they emerge on anoutgoing tide, mate and lay eggs, and arewashed out to sea on the incoming tide. Eggsof the marine species are laid singly in rock

Evolutionary Relationships of Telmat ogeton-r- t-aswws i« 57

crevices, and larvae feed by grazing on the, thin film of algae growing on the rock sur-

faces and on macroscopic green and redalgae. Larvae build silky tubes from theirsalivary gland secretion; pupation occurs inreinforced tubes . The tubes protect larvaeand pupae of the marine species from waveaction at high tide and predation and des-iccation at low tide, and thus allow larvaeand pupae of the freshwater species to livein torrential streams and waterfalls in theHawaiian Islands .

In 1971, Hampton Carson discovered theHawaiian Telmatogeton as a material suit-able for chromosome analysis (personalcommunication). He found the giant poly-tene chromosomes of the larval salivaryglands to be well banded and likely to giveinformation relative to the evolution of thefreshwater species. This proved to be true ,for Newman (1977) established a standardband map and proposed a pro visional set ofevolutionary relationships for the Hawaiianfreshwater Telmatogeton.

All the species examined to date havepolytene chromosomes suitable for detailedband ana lysis. Each species has six longchromosome ' arms with well-developedbands and a single unbanded short arm . Thesix long arms were designated by the lettersA-F and the short arm by the letter G. Asequence of bands within arms A-F com-mon to most of the Hawaiian freshwaterspecies was chosen as the Telmatogetonstandard (Newman 1977). The chromosomearms of most of the species examined havethe standard sequence or differ from it byparacentric inversions . A paracentric inver-sion is a chromosomal mutation in which aseries of bands that does not contain the cen-tromere is rotated 1800 • The specific breakpoints of the inversion may be determined bycomparing the inverted and standard bandsequences. Inversions are designated by giv-ing the arm letter of the inversion with a sub-script arabic number. An inversion may behomozygous in all animals of a populationor species; that is, it is fixed or it may existalong with the standard as a polymorphism.In some cases, the rearrangement of bands ina portion or even an entire arm may be socomplicated by multiple inversions that it

cannot be described in terms of simple in-versions. These are referred to as complexportions of an arm or complex arms .

Chromosome numbers in the Telmato-geton and Paraclunio range from n = 7 ton = 3/4 (Newman 1977). It has been pro-posed that the primitive number is n = 7 andthat the other numbers are derived by cen-tric fusions. Each centric fusion reduces thechromosome number by 1. Telmatogetonhirtus (n = 3/4) has an XY1Y z sex chromo-some system that may be seen in both polyteneand nonpolytene chromosomes . Telmatogetonabnormis (n = 4) has an XY chromosomesystem that may be seen only in the polytenechromosomes. Other species do not have dif-ferentiated sex chromosomes.

Inversion sequences have been used togain insight into possible evolutionary rela-tionships among species. Species that shareinversions are more closely related thanspecies that do not share inversions . Carson(reviewed in Carson and Yoon 1982) usedthis type of analysis to determine the evolu-tionary relationships of over 100 species ofHawaiian Drosophila . Similar techniques areused here to propose a set of relationships inwhich there were at least two separate in-vasions of marine Telmatogeton into freshwater in the Hawaiian Islands .

MATERIALS AND METHODS

The giant banded polytene chromosomeswere examined from cells of the salivarygland of fourth instar larvae fixed in aceticalcohol and stained in lactoacetic orcein.Nonpolytene chromosomes for the determi-nation of chromosome numbers were derivedfrom lactoacetic orcein squashes of testesand other developing larval tissues (details inNewman 1977).

RESULTS

A list of the described species of Telmato-geton and Paraclunio is given in Table 1.Band sequence and chromosome numberdata for all the species examined to date aregiven in Table 2. Included are data for an un-

58

SPECIES

Te/matogeton, marineT. japonieus TokunagaT. paeifieus TokunagaT. pusillum Edward sT. maeswaini WirthT. /atipennis WirthT. sancti-pauli SchinerT. minor KiefferT. australi eus WomersleyT. antipodensis Sublette and WirthT. mortoni LeaderT. simplicipes EdwardsT. troeanteratum EdwardsT. at/antieum OliveiraT. nanum Oliveira

Te/matogeton , Hawaiian freshwaterT. hir tus WirthT. abnormis TerryTi fluviatilis WirthT. williams i WirthT. torrent ico/a Terry

Parac/unio, marineP. tri/obatus KiefferP. spinosus HashimotoP. a/askensis Coquillett

PACIFIC SCIENCE, Volume 42, January/Jul y 1988

TABLE I

S PECIES OF Te/matogeton AND Parac/unio

GEOGRAPHIC DISTRIBUTION

Japan, Hawaii,* Florida, Australia, Baltic Sea*Japan, Hawaii*Marquesas, Mariana, MicronesiaCalifornia*Revillagigedo Island s (Pacific Ocean , Mexico)South Africa, St. Paul Island (Ind ian Ocean)South AfricaAustraliaAntipodes Islands (New Zealand)New ZealandSouth America (Ancud , Chile)South America (Ancud , Chile)South AmericaSouth America

Kauai*Kauai,* OahuOahu *OahuMolokai,* Mau i,* Hawaii*

Washington ,* Oregon,* California*Californ ia*Alaska,* British Columbia,* Washington,* Oregon ,* California*

List of species from: Wirth (1947,1959), Hashimoto (1976), Sublette and Wirth (1980). Species moved into the genus Telmatogetonby Sublette and Wirth (1980) are not included in this table.

• Chro moso mes examined.

described species from east Maui designatedas T. new species- I (T. n. sp.-I). Most of thespecies were discussed in Newman (1977);new data are reported here for T. japonicus,T. macswaini , the three Paraclunio species,and an unidentified Telmatogeton speciesfrom southeast Maui. Adults, required forspecies identification, were not collected forlarvae of the southeast Maui population.

The six chromosome arms of the marinespecies Telmatogeton pacificus are totallycomplex relative to the Telmatogetonstandard and thus it does not appear to bedirectly related to the other species. Thefreshwater species T. hirtus is largely com-plex relative to the Telmatogeton standard.Arm A differs from the standard by two fixedinversions, and the remaining arms are com-plex but do contain short segments that canbe matched to the standard.

Examples of shared inversions appear inFigures I and 2. Populations of Telmato-

geton japonicus, T. macswaini, and T. abnor-mis are fixed for two inversions (C3,4 ) inwhich inversion C4 is included within thelimits of C3 (Figure I). Two of the four breakpoints appear to be at the same site in thepolytene chromosome. Arm C of T. n. sp.-lis complex but has one of the C3 break points.Telmatogeton japonicus and T. n. sp.-I shareinversion E2 (Figure 2). In addition, eachspecies has another inversion with one breakpoint within the limits of E2 . The proposedderivation of these chromosomes is discussedbelow.

Telmatogeton japonicus and T. n. sp.- lshare fixed inversion B5 as well as E2 . InT. japonicus, arm F is standard, arms C andE differ from standard by three and twoinversions, respectively, arms A and B arepartly standard, and arm D is complex. ForT. n. sp.- l , arms B, E, and F may be derivedfrom the standard while arms A, C, and 0are at least partl y complex.

trlTABLE 2

60 PACIFIC SCIENCE, Volume 42, January/Jul y 1988

T mecswetnt

T abnormis

T japonicus

T new species-1

FIGURE I. Chromosome arm C from four species of Telmat ogeton . Inver sion C 3.4 is shared by T. macswaini, T.abnormis, and T. japonicus. In addition, T. macswaini has inversion C6 and T. japon icus has inversion C 7 . Telmato-geton new species-I has the C3 break point proximal to the centromere. Centromeres are to the right for each chrom o-some arm.

The chromosomes of the remaining fresh-water species have, or are close to, thestandard sequence. Telmatogeton torrenticolais a species complex composed of five siblingspecies that differ by chromosome numberand fixed inversions. The west Maui popula-tions contain the standard band sequencesfor all chromosome arms and a chromosomenumber of n = 7. Populations to the east(on east Maui and the Kohala Mountainsand Mauna Kea on Hawaii) accumulate

fixed inversions and have decreased chromo-some numbers. To the west, populations ofT. torrenticola on Molokai also differ fromthose of west Maui by fixed inversions andreduced chromosome number (deta ils inTable 2). Telmatogeton flu viatilis and the un-identified species from southeast Maui eachdiffer from the standard by only one fixedinversion, and T. abnormis differs from thestandard by three inversions.

The North American marine species

Evolutionary Relationships of Telmatogeton-s-tsevossi«

Intermediate (Ez)

Standard E

T. new spec ies-1 (EZ•3 )

T. japonicus (Ez,4 )

4

61

FIG URE 2. Evolution of chromosome arm E in T. new species- I and T. japonicus. The standard sequence (lowerleft) is considered to be primitive . Inversion E2 is intermediate (upper left; interstitial segment of photograph inlower left cut and inverted to show inversion E2 • Te/matogeton new species-I shows inversion E3 and break points ofE4 (upp er right) , and T. japonicus shows inversion E4 and break points of E3 (lower right) . Centromeres are to theright in each photograph. .

Te/matogeton macswaini differs from thestandard by six inversions in arm s C, D, andF. It shares the standard sequence in arms A,B, and E with other species, inversions C3 , 4with T. abnormis and T.japonicus, and inver-sion D 2 with T. torrentico /a from west Ma ui.

Of the three Parac/unio species, P. tri/obatusis closest to the standard. It shares thestandard sequence in arms A and E withother species. Arms 0 and F are partlystandard, and arms Band C are completelycomplex. Parac/unio spinosus shares thestandard sequence of arm A as well as por-tions of arms 0 and E with other species.Arm F differs from the standard by twoinversions, and arm s Band C are complex .Inversion F9 is shared with T. macswaini.

Parac/unio a/ask ensis is divided into atleast two dist inct chromosomal forms. Anorthern form extends to the north from atleast Yakutat, Alaska , through Moss Land-

ing, Californ ia, to the south; and a southernform extends from Monterey, California,through Gaviota, California. The two formsare separated by about 30 km; hybrids be-tween the forms have not been found . Onlyportions of arms A, B, 0 , E, and F of thenorthern form fit the standard sequence; theremaining segments of these arms are com-plex. The two forms differ from each otherby a single fixed inversion in arm s A and 0 ,arms B, E, and F have the same sequences,and there are few similarities between thetwo form s for arm C.

DISCUSSION

Previous studies on the Hawaiian Telma-togeton indicated a single emergence fromthe marine to freshwater environments.Wirth (1947) proposed a single, unbranched

62 PACIFIC SCIENCE, Volume 42, Janu ary/Jul y 1988

pattern of evolution with a T. japon icus-likespeciesas the marine ancestor. The freshwaterspecies evolved in the orde r: T. abnormis,T. flu viatilis, T. williamsi, T. torrentico/a, andT. hirtus. His analysis was based primarily ondifferences in the morphology of tar sal clawsin adult males.

Chromosome data (Newman 1981) alsoindicated Te/mat ogeton jap onicus as thelikely marine ancestor to the freshwaterspecies in that it shares several band sequenceswith the freshwater standard. Complete datafor T. jap onicus were not available at thetime because of poor material. However,T. n. sp.-l was proposed as the first fresh-water species because it shared the ancestralchromosome number of n = 7 and at leastinversion B5 with T. japonicus. The standardsequence, found in all arms of T. torrentico/a

from west Maui , was then derived fromthe T. n. sp.-l sequences by inversions. Theother forms of the T. torrentico/a complexand the remaining freshwater species werethen derived from the standard sequence.

Better chromosomal data from Te/-matogeton japonicus and an analysis of thechromosomes of the North American Te/-matogeton and Paraclunio species now sug-gest that the Te/matogeton standard bandsequences are for the most part ancestralrather than derived for the freshwater speciesother than T. n. sp.-l . At least two separateemergences from marine to fresh water oc-curred (Figure 3). One was from T. jap onicusto T. n. sp.-l , and the other was througha hypothetical ancestor to T. abnormis andT. torrentico/a from west Maui .

It is proposed that Te/matogeton japonicus

T. f1uviatilisOahun =7C51 inv. to std.

Unidentif ied speciesSoutheast M auin ~7

Fa1 inv. to std.

T.abnormisKauain =4C3,. ; B2; (0 +)3 invs. to std .

T. macswain iNort h Amer ican =5C3,. ; Ca; 0 2; Da; Fg6 invs. to std.

T. torrentico/a complex

Haw aii; M auna Kean ~ 4 0 3.5; Fa

Hawaii; Kohala Mtns .n ~ 5 0 5; Fa

IEast Mau in ~ 6 0 5

West Maui~ n ~ 7

gen us standa rd (0 2/ + )

tIMol okai

n ~ 4 A2; 0 4; E,

Marine hypotheticalMaj or islandsn ~7

C3,. ; 0 2/ +

T. new species-jEast Mauin =7B5; E2.3; F2.3.4;complex in portions

of A. C. °Fresh wate r

Marine

T. japonicusMajor islandsn =785; E2.4; C3,. ; C7complex in portion sot A, B, D

FIGURE 3. Evolu tionary scheme for Telmatogeton species from the Hawaiian Islands and North America . Inter-mediate chro moso mal forms in circles; freshwater species above and marine species below horizontal line. See textfor complete explanation .

Evolutionary Relationships of Telmatogeton-NEwMAN 63

and T. n. sp.-I had a common ancestor inone emergence from marine to fresh watersince they share inversions Ez and Bs. Arm E

, of the ancestor was standard. Inversion Ezwas fixed in an intermediate species; then in-version E3 was fixed in T. n. sp .-I and inver-sion E4 was fixed in T. japonicus (Figure 2).Inversion Bs , also present in the inter-mediate species , remained fixed in bothspecies . Inversions C 3 , 4 were retained fromthe common ancestor in the evolution ofT. japonicus, and additional inversions in theevolution of arm C of T. n. sp.-I renderedthat arm complex except for the C3 breakpoint (Figure 1). Both species retained thechromosome number ofn = 7.

The second invasion into fresh water in-volves Telmatogeton macswaini, T. abnormis,and T. torrenticola from west Maui since thechromosomes of these species are close to theTelmatogeton standard sequence. A marinehypothetical species is proposed as an ances-tor to the remaining freshwater species. Ithas the Telmatogeton standard band sequencewith the exception of fixed inversions C3 •4and it is polymorphic for inversion D z. Inthe line from the marine hypothetical toT. abnormis, inversions C 3 , 4 are retained andinversion Bz is fixed. The complete Telmato-geton standard is derived from the marinehypothetical by the inversion of C 3 , 4 to thestandard . The Dz pol ymorphism and chro-mosome number of n = 7 are retained in theevolution of T. torrenticola from west Maui.The chromosome numbers are reduced bycentric fusions in the other sibling species ofT. torrenticola. Telmatogeton fluviatilis andthe unidentified species from southeast Mauiare deri ved separately from an ancestor withthe standard band sequences. The place ofT. macswaini in the evolutionary scheme isunclear. It ma y have been deri ved from aform like the hypothetical ancestor with thefixation of four inversions, the retention ofC3, 4 , and the reduction in chromosome num-ber from n = 7 to n = 5 by centric fusions .Telmatogeton hirtus and T. pacificus are notconsidered here since their chromosomes areso complex.

The relationships of the Paraclunio speciesto the Telmatogeton and even among each

other is no t clear. Paraclunio trilobatus andP. spinosus are closer to the Telmatogetonstandard than is P. alaskensis. In P. tri-lobatus, two of the six arms are standard, andin P. spinosus, one arm is standard. Para-clunio spinosus and T. macswaini share inver-sion Fg • Placement of the Paraclunio speciesinto the Telmatogeton ph ylogeny must awaitbetter analysis of the complex chromosomearms.

It is not surprising that Telmatogetonfound its way into freshwater streams oreven that multiple invasions to fresh wateroccurred. Wirth (1947, 1959) proposed thatchironomids, which are adapted to marineenvironments, evolved from freshwater spe-cies because of the great competition found inthe freshwater environment. In the HawaiianIslands, where there was a lack of competi-tion in fresh water, species of Telmatogetonmoved back into fresh water. Limited vagilityand reduction of population size or extinc-tion of local populations caused by the severeenvironment may have favored the evolutionof Telmatogeton.

At least some species of Telmatogeton andParaclunio have retained their tie to freshwater. Wirth (1947) collected T. japonicus inan area of reduced salinity in the HawaiianIslands. Tokunaga (1935) reported the devel-opment of T . japonicus from lar vae to adultsin fresh water in the laboratory. Populationsof P. alaskensis were ob served by the authorto live in freshwater seepages on rock wallssubject to saltwater only at high tide . Parkin-son and Ring (1983) reported that larvae ofP. alaskensis are osmoregulators and arethus able to adjust the ionic concentration oftheir hemolymph when living in fresh water.

The chro mosome analysis of Telmatogetonand Paraclunio is incomplete. New specie s ofTelmatogeton ar e still being disco vered bothin the Hawaiian Islands and other parts ofthe world (Sublette and Wirth 1980) andmany of the described species remain to beanalyzed (Table I). However , the new dataand an alysis pre sented here brings the evolu-tionary scheme of the freshwater Telmato-geton more in line with the morphologicalana lysis of Wirth (1947) ; that is, T. abnormisis placed close to one point of emergence

64 PACIFIC SCIENCE, Volume 42, January/Jul y 1988

from the marine environment. New datafrom both morphology and cytogenetics arerequired for a better understanding of evolu-tion in the genus Telmatogeton.

ACKNOWLEDGMENTS

My thanks to Hampton L. Carson for in-troducing me to cytogenetics many yearsago , to D. Elmo Hardy and Kenneth Kane-shiro for their help during field trips in theHawaiian Islands, and to Jill Turner forreading and correcting the manuscript.

LITERATURE CITED

CARSON, H. L., and J. S. YOON. 1982. Ge-netics and evolution of the Hawaiian Dro-sophila. Pages 297-344 in M. Ashburner,H. L. Carson, and J. N. Thompson, Jr. ,eds. The genetics and biology of Droso-phila. Vo!. 3b. Academic Press, London.

HASHIMOTO, H. 1976. Nonbiting midges ofmarine habitats (Diptera: Chironomidae).Pages 377-414 in L. Cheng, ed. Marineinsects. Elsevier, New York.

KRONBERG, I. 1986. Riesenchromosomen undArtareal einer baltischen Telmatogeton-Art (Diptera: Chironomidae: Telmatoge-toninae) Z. zoo!. Syst. Evolur.-forsch. 24:190-197.

MORLEY, R. L., and R. A. RING. 1972. Theintertidal Chironomidae (Diptera) of BritishColumbia II . Life history and populationdynamics. Can. Entomo!. 104: 1099-1121 .

NEWMAN, L. J. 1977. Chromosomal evolu-

tion of the Hawaiian Telmatogeton (Chiro-nomidae, Diptera). Chromosoma (Ber!.)64: 349-369.

- - -. 1981. Evolution of gnats of thegenus Telmatogeton. Pages 467-470 inD. Mueller-Dombois, K. W. Bridges, andH. L. Carson, eds. Island ecosystems: Bio-logical organization in selected Hawaiiancommunities. Hutchinson-Ross, Strouds-burg, Pa.

PARKINSON, A., and R. A. RING. 1983. Osmo-regulation and respiration in a marinechironomid larva , Paraclunio alaskensisCoquillett (Diptera, Chironomidae). Can.J. Zoo!' 61: 1937-1943.

ROBLES, C. 1982. Distribution and predationin an assemblage of herbivorous Dipteraand algae on rocky shores. Oecologia(Ber!.) 54: 23-31.

SUBLETTE, J. E., and W. W. WIRTH. 1980.The Chironomidae and Ceratopogonidae(Diptera) of New Zealand's subantarcticislands. New Zealand J. Zoo!. 7:299-378.

TOKUNAGA, M. 1935. Chironomidae fromJapan (Diptera), IV. The early stages ofa marine midge, Telmatogeton japonicusTokunaga. Philipp . J. Sci. 57:491-511.

WIRTH, W. W. 1947. A review of the genusTelmatogeton Schiner , with descriptionsof three new Hawaiian species (Diptera:Tendipedidae). Proc . Hawn. Entomol.Soc. 13: 143-191.

---. 1959. A revision of the Clunioninemidges with descriptions of a new genusand four new species (Diptera: Tendipe-didae). Univ. California Pub. Entomol.8: 151-182.