Identification of Asteroid Genera With Species Capable of Larval Cloning

10 Pages·2003·4 MB·English

by  K Emily Knott

Reference: Biol. Bull. 204: 246-255. (June 2003)
200.1 Marine Biological Laboratory
Identification of Asteroid Genera With Species
Capable of Larval Cloning
K. EMILY KNOTT1 *, ELIZABETH J. BALSER2, WILLIAM B. JAECKLE2.
AND GREGORY A. WRAY't
1 Department ofEcology and Evolution. State University ofNew York, Stony Brook, New York 11794-5245;
and 2Department ofBiology, Illinois Wesleyan University, Bloomington, Illinois 61702-2900
Abstract. Asexual reproduction in larvae, larval cloning, complexity has been recognized in the diverse developmen-
is a recently recognized component of the complex life tal modes exhibited by asteroid echinoderms (sea stars).
histories of asteroids. We compare DNA sequences of mi- Nearly all asteroids reproduce sexually and have complex
tochondrial tRNA genes (Ala. Leu, Asn. Pro, and Gin) from life cycles in which larval stages, having very different
larvae in the process of cloning collected in the field with morphologies and habits from the adults, alternate with
sequences from adults ofknown species in order to identify adult stages (Mortensen, 1921; Hyman. 1955; Chia and
asteroid taxa capable ofcloning. Neighbor-joining analysis Walker, 1991 ). Asexual reproduction by adults is prevalent
identified four distinct groups of larvae, each having no, or in some asteroid groups (e.g.. l.inckiu, Coscinasterias) and
very little, sequence divergence (/> distances ranging from supplements the product ofsexual reproduction by increas-
0.00000 to 0.02589); thus, we conclude that each larval ing the number ofindividuals derived from a given lineage.
group most likely represents a single species. These field- Asexual reproduction by larvae, larval cloning, is poorly
collected larvae cannot be identified to species with cer- understood, including which species are capable of it and
ltaairnvtayl,gbruotutphseicnldoisceataesssegmenbelraigceoorffkanmiolwinaltiadxeantwiittyh. tWhee fcoaunr washtaetroirdosl.e it might play in the ecology and evolution of
assign two ofthe larval groups discerned here to the genera Three distinct modes of larval cloning have been ob-
Luiclia and Oreaster and another two to the family Ophidi- served in planktotrophic asteroid larvae collected from the
asteridae. This study is the first to identify field-collected field and reared in the laboratory (Bosch, 1988; Bosch ctul.,
cloning asteroid larvae, and provides evidence that larval 1989: Rao ct ul.. 1993; Jaeckle. 1994; Vickery and Mc-
cloning is phylogenetically widespread within the Asteroidea. Clintock, 2000; Kitazawa and Komatsu, 2001). These
Additionally, we note that cloning occurs regularly and in modes paratomy of the posterolateral arms, autotomiza-
emlaumrlpvthaialpslcielzoiwnnaigyngsthiewninttehheiednefcsooprlecofiugerysthaetnrhdaitnevvaeorsletuitcgiaaotpniaobonlfeoafsottfheercolrioodnlsie.ngo,f tainodn aorfmthteippsreosrahlarelobien,caonmdmbounddainpgerfiroodmofthdeedliafrfvealrenbtoiday-
tion of larval tissues that are then redifferentiated in the
Introduction clone (see Jaeckle. 1994. for details). Larval cloning in
benthic. brooded, and pelagic lecithotrophic larvae has not
With the discovery oflarval cloning (Bosch, 1988, 1992; been observed but may occur through some as yet unrec-
Bosch c/ ,//.. 1989; Rao ct ul.. 1993; Jaeckle. 1994). new oanized process. Clones are able to develop to and through
metamorphosis and may themselves exhibit larval cloning
Received IS Ounher 2002; accepted 13 March 2003. (EJB and WBJ, pers. obs.; Vickery and McClintock, 2000;
* To whom correspondence should be addressed. Present address: De- Kitazawa and Komatsu. 2001). However, it is not known
partment of Biological and Environmental Sciences, University ofJyvas- whetherjuveniles derived from cloned larvae will develop
kylta,Cu4r0r0e1n4t Jaydvdarsesksy:la,DeFpianrltanmde.ntEmoafilB:[email protected]. Durham, to sexual maturity, or if larval cloning has fitness conse-
NC 27708-0338. quences for either the primary or cloned larvae. Larval
246
ASTEROIDS CAPABLE OF LARVAL CLONING 247
cloning could possibly have a significant impact on asteroid culturing methods, we have used DNA sequence similarity
life history by altering such parameters as dispersal, number to identify field-collected cloning asteroid larvae. DNA
of individuals, or fitness, thus emphasizing the need to sequencing techniques are universally known and easily
identify those asteroid species capable of larval cloning. implemented in the laboratory for a quick assessment of
Asteroid larvae in the process of cloning have been potential larval identity. Identity then can be verified with
collected from portions ofthe tropical and subtropical west- more time-consuming laboratory culturing techniques.
ern Atlantic Ocean (Jaeckle, 1994), including the Sargasso
Sea (Bosch. 1988; Bosch ct al., 1989). and from the Bay of Materials and Methods
Bengal (Raoetal.. 1993). These larvaecan be very common
in collections, constituting from 10% to 90% ofthe asteroid We investigated the identity of field-collected cloning
DNA
larvae present (Bosch, 1988; Bosch ct al., 1989; Jaeckle, asteroid larvae by comparing the sequences of five
1994; EJB and WBJ, pers. obs.). However, field-collected mitochondria! tRNA genes (Ala, Leu. Asn. Pro, and Gin)
cloning larvae have not been identified specifically. Ini- from the larvae to complementary sequences from adults of
tially, cloning larvae were thought to be restricted to species known species. The suitability of this gene region for spe-
in the genus Luidia, which have bipinnaria larvae that lack cies identification was initially assessed by comparing se-
a brachiolarcomplex and are, in some Luidia species, quite quences obtained from a single known larva of Luidia
large (Wilson, 1978; Domanski, 1984; Bosch et al., 1989). clathmta to sequences from related, known adult asteroids.
But Bosch (1992) and Jaeckle (1994) showed that larval Comparison of sequences from the L. clathrata larva, a L
cloning is not taxonomically restricted when they reported clathmta adult from a different locality, other Luidia spe-
larval cloning in brachiolaria larvae, which are common to cies, and species from the closely related genera Astro-
all asteroid orders except the Paxillosida (to which Luidia pecten and Ctenodiscits are shown in Table 1. The se-
belongs). quences obtained for the L. clathrata larva and adult are
Cloning larvae have been observed in laboratory cultures identical. However, significant nucleotide changes are ob-
as well. Previous laboratory studies have noted larval clon- served between L. clathratii (larva and adult) and other
ing in members of the Paxillosida and Forcipulatida (EJB Luidia species, as well as between L. clathrata (larva and
and WBJ. pers. obs.; Vickery and McClintock, 2000; adult) and species in other genera, which are reflected in
Kitazawa and Komatsu, 2001). These occurrences, in spe- genetic distances among species (Table 2).
cies found in the north Pacific, indicate that larval cloning Larvae used in our comparisons were collected from the
may be more widely distributed geographically than previ- tropical and subtropical western Atlantic Ocean, specificaWlly
ously recognized. However, field-collected cloning larvae from offthe western shore ofBarbados (31 2" N, 59 4" )
have not been reported in these areas, and whether larval and from the Gulf Stream off the eastern shore of Florida
cloning occurs naturally in these species (i.e., outside the (27.3 N. 79.6 W). by EJB and WBJ. Cloning larvae have
laboratory) is not known. been collected consistently and in large numbers at the Gulf
Our goal is to identify asteroid larvae capable ofcloning Stream site (EJB and WBJ, pers. obs.). Individual larvae
by comparing the DNA sequences of unknown, field-col- were scored for the presence and type ofcloning exhibited.
lected cloning larvae and known adult species. Because Most of the larvae were cloning by paratomy, but one was
most asteroid larvae are morphologically similar, field-col- cloning by autotomy ofthepreoral lobe. Thosethat were not
lected larvae can rarely be identified to family level, much cloning (14 of 65) were similar to cloning larvae found in
less to genus or species, by visual inspection of morpho- the same or in other collections, and so were assumed to
logical characteristics. Indirect identification ofasteroid lar- have the ability to clone (Fig. 1 ). All larvae were preserved
vae based on correlations with geographical distributions of in 95% EtOH and shipped to SUNY Stony Brook for
adults is unlikely because larvae may have great dispersal processing.
potential and do not necessarily remain close to their pa- The five mitochondria! tRNA genes ofinterest (Ala, Leu,
rental population (Thorson, 1961; Strathmann, 1974; Schel- Asn, Pro, and Gin) were amplified from individual larvae
tema, 1986). Morphological identification ofjuveniles after using the polymerase chain reaction (PCR) and published
metamorphosis is possible, but not always practical or de- echinoderm-specific primers (Smith et ai, 1993; Hart etal.,
pendable. Asteroid larvae are sensitive to laboratory cultur- 1997). A typical total genomic DNA extractionwas avoided
ing conditions (Strathmann. 1987). can take weeks to because these extraction techniques often require a large
develop (e.g., Komatsu et al., 1991). and can delay meta- amount of starting material. Instead, the entire larval body
morphosis for several months ifa suitable settlement cue is (first air-dried to remove traces of EtOH) was used in PCR
not found (Pechenik, 1990). Often, laboratory cultures die as the DNA template (Medeiros-Bergen et al.. 1995). The
before the larvae reach metamorphosis or before juveniles thermocycling conditions of PCR are severe enough to
are large enough to be identified. As an alternative to disrupt the larval cells, releasing their DNA. Amplifications
248 K. E. KNOTT ET AL
Table 1
Alignmentof tRNA sequences (Ala, Leu, Asn, Pro, andGin} showing complete sequence identity bet\veen Luidiaclathrata adult andlan-a; sequence
differences benveen Luidia clathrata andother Luidiaspecies (*), otherclosely relatedgenera (+), orboth (") are marked
Ala
Luidiaclathrata -GTGAATTTAGTTTAAAA-GAA- -AAAACCTTTGATTTGCATTCAAAAA-- --A-TTTAGGT--TTAAGACCTAAAATTTACA-
L clathrata larva -GTGAATTTAGTTTAAAA-GAA--AAAACCTTTGATTTGCATTCAAAAA --A-TTTAGGT--TTAAGACCTAAAATTTACA-
L. magellanica -GTGAATTTAGTTTAACA-GAA- -AAAACATTTGATTTGCACTCAAACA-- --A-TTTAGGT--TTAAACCCTAAAGTTTACA-
L. foliolata -GTGAATTTAATTTAAAA-GAA--AAAATATTTGATTTGCATTCAAACA-- --A-TTTAGGT--TTAAAGCCTMAGTTCACA-
L altemata -GTGAATTCAGTTTAAGA-GAA--AAAACTTTTGACTTGCATTCAAAAA-- --A-TTTAGGT--TTAACTCCTAAAATTTACA-
Astropecten -GTGAATTTAGTTTAAAA-GAC--AAAACATTTGATTTGCATTTAAAAA-- --A-TCCAGGT--TTAATTCCTGGAATTCACA-
Ctenodiscus -GTGAATTTAGTTTAAAA-GAT--AAAACA'TT+TA*ATTTGCATTT+AA*AAA-- -C+-+TT+CAAGT -TTAACCCCTG+A+AA+TC"CACA-
* 4-
Leu
Luidiaclathrata -GCTAAAATAGCAAAGTG-GTA--AATGCTGTAGATTTAGGTTCTATTA-- --T-CAAAGGTTCAAATCCTTTTTTTAGTT--
L. clathrata larva -GCTAAAATAGCAAAGTG-GTA--AATGCTGTAGATTTAGGTTCTATTA-- --T CAAAGGTTCAAATCCTTTTTTTAGTT--
L magellanica -GCTAGAATAGCAAAGGG-GTA--AATGCAATAGATTTAGGATTTATTA-- --T CAAAGGTTCAATTCC-TTTTTTAGTT--
L.foliolata -GCTAAAATAGCAAAGTG-GTA- -AATGCAATAGATTTAGGATTTATTA- - --C-CAAAGGTTCAATTCCTTTTTTTAGTT--
L altemata -ACTTAGGTAGCAAAGCG-GTA--AATGCGGTAGATTTAGGATCTATTA - T-CAGGGGTTCGATTCTCTTCCTTAGTT--
Astropecten -GTTAGAATAGCAAAGGG-GAA--AATGCAATAGATTTAGGATCTGTCA-- --T-CAAGAGTTCGAGTCTCTTTTCTAGTT- -
Ctenodiscus -ACTGAGGTAGCAAAGTG-GTG--AATGCGGCAGATTTAGGATTTGTTA-- --T CAAGGGTTCTAATCCCTTTCTTAGTT--
+ - ++ "+ '++ "+ "+
Asn
Luidia clathrata -TGGGTTGTAGCCTAGT--GGA--AAGGCAACTGGCCGTTAACCAGGAG-- *-ATAACAAGATCAATACTTGTCAACTCAG--
L. clathrata larva -TGGGTTGTAGCCTAGT--GGA--AAGGCAACTGGCCGTTAACCAGGAG-- --ATAACAAGATCAATACTTGTCAACTCAG--
L. magellanicii -TGGGTTGTAGCCTAGC--GGA--AAGGCAACTGGCCGTTAACCAGGAG- - --ATAACAAGATCAATACTTGTCAACTCAG--
L.foliolata TGGGTTGTAGCCTAGT- -GGA--AAGGCAACTGGCCGTTAACCAGGAG-- --ATAACAAGATCAATACTTGTCAACTCAG--
L lillt-mata -TGGGTTGTAGCCTAGT--GGA--AAGGCAACTGGCCGTTAACCAGGAG-- --ATAACAAGATCAATACTTGTCAACTCAG--
Astropecten -TGGGTTGTAGCCTAAT--GGA- -AAGGCAATTGGCCGTTAACCAGGAG-- --ATAGTAAGATCAATACTTACCAACTCAG--
Ctenodiscus -TGGGTTGTAGCCTAGT--GGA- -AAGGCAATTGGCCGTTAACCAGGAG-- - -ATAACAAGATCAATACTTGTCAACTCAG
+* + ++ ++
Pro
Luidiaclathrata -CAGAGAATAGTTTAATT-TAG--AGAATTGTAACTTTGGGAGTTATTG-- --G-TACAAATATA-GAGTTTTGTTTCTCTGA-
L. clathrata larva -CAGAGAATAGTTTAATT-TAG- -AGAATTGTAACTTTGGGAGTTATTG-- --G-TACAAATATA-GAGTTTTGTTTCTCTGA-
L. magellanica -CA?AGAATAGTTTAATT-TAAA-AGAATTGTAACTTTGGGAGTTATTG-- --G-TGCAAATGTA AAGTTTTGTTTCTCTGA-
L.foliolata -CAGAGAATAGTTTAACA-TAAA-AGAATTATAACTTTGGGAGTTATAG-- --G-TGCAAATGTA-GAGTTTTGTCTCTCTGA-
L. ultemata -CAGAGAATAGTTTAGTT-T AGAATAATAACTTTGGGAGTTATTG-- --G-TGCAAATATA-GAGTTTTGTTTCTCTGA-
Astropecten -CAGAAAATAGTTTAATT-- - -AGAATAATAACTTTGGGAGTTATTG-- --G-TGTAAATATA-GAGTTTTATTTTTCTGA-
Ctenodiscus -CAGGAAATAGTTTAATA-- --AGAATGATAGCTTTGGGAGTTGTTA-- --G-TGTAAATATG-GAATTTTACTTTTCTGA-
++ .-_(_--. _|_ + + . + ++. 4.
Gin
Luidiaclulhrttta -TAGAAAGTAGTATAAT--GGA- -ATTACAAAGATCTTTGACTTCTTAA-- --A-TATAAGTTCAATCCTTATCTTTCTAA--
L. clathrata larva -TAGAAAGTAGTATAAT--GGA- -ATTACAAAGATCTTTGACTTCTTAA-- --A-TATAAGTTCAATCCTTATCTTTCTAA--
L. magellanica -TAGAAAGTAGTATAAA--GGT--ATTACAAAGATCTTTGACTTCTTAA- -A CATAAGTTCAATTCTTATCTTTCTAA--
L. foliolata -TAGAAAGTAGTATAAA--GGC--ATTACAAAGATCTTTGACTTCTTAA-- --A-CATAAGTTCAATTCTTATCTTTCTAA- -
L. altemata -TAGAAAGTAGTATAGGC-GGA--ATTACAAAGACCTTTGACCTCTTAA-- --A-CATAAGTTCAACTCTTGTCTTTCTAA--
Astropecten -TAGAAAGTAGTATAAT--GGT--AAAACAAAGAACTTTGACTTCTTTA-- --A-TATAAGTTCAATTCTTATCTTTCTAA--
Clenodiscus -TAGAAAGTAGTATAAC--GGC--AATACATAGAACTTTGATTTCTTAA-- - C-TACAAGTTCAATTCTTGTCTTTCTAA--
123456
Table 2
Genetic distancescalculatedusing methodsforuncorrected (p) distance bet\veen tRNA genesfrom a known Luidia clathrata larva, otherLuidia
species andspeciesfrom closely relatedgenera
1. Luidia clulhralu
2. Luidia clathi\iut larva 0.00000
3. Luidia magellanica 0.06422 0.06422
4. Luidiafoliolata 0.06948 0.06948 0.04450
5. Luidia alternant 0.09243 0.09243 0.10482 0.11230
6. Astropecten 0.12597 0.12597 0.12657 0.13699 0.14601
7. Ctenodiscus 0.14561 0.14561 0.16100 0.15135 0.14592 0.14566
ASTEROIDS CAPABLE OF LARVAL CLONING 249
Figure 1. Representative asteroid larvaewithclonescollectedfrom plankton samplestaken offthe eastern
coast ofFlorida. Obvious morphological characters such as color and arm length appear to be labile and are
unreliable as taxonomiccharacters. Definitive morphotypic charactersdistinguishing species have notyetbeen
identified. (A-D) Brachiolariae included in larval group 1. (E) Bipinnaria oflarval group 2. which includes at
leastoneLuidiaspecies. (F)Bipinnariafromlarvalgroup3/4. Asterisksindicatelarvalclones. Scalebars = 150
were successful for 44 out of 65 (68%) larvae collected. Miniprep purification kit (Promega), sequenced in forward
Amplification reactions were carried out in 25-juJ volumes and reverse directions using vector sequence primers [Ml3
of a standard reaction mix with Taq DNA polymerase (20) Ml3 (rev)] and dye-terminator sequencing reaction
(GIBCO Life Technologies) using a MJ Research PTC-200 mix. and analyzed with an ABI 373 automated sequencer
thermocycler. All samples were purified in 2% NuSieve (PE Applied Biosystems). Sequence data from both strands
agarose (FMC BioProducts) and gel extracted (QIAGEN or were combined and edited with Sequencher 3.0 (Gene
GIBCO Life Technologies kits). Purified samples were Codes Corporation. 1995) and aligned by eye in accordance
chemically transformed (Brown, 1991) into XLI Blue with established tRNA alignment ofothertaxa based on the
(Stratagene) competent cells using pGEM-T vector (Pro- molecular structure of tRNAs (Sprinzl et ai, 1998). Larval
mega). Cloned samples were purified with Wizard Plus sequences were then aligned to similarly aligned sequences
250 K. E. K.NOTT ET AL
of the same genes from known adult asteroid species (Hi- species likely to be capableoflarval cloning. Representative
meno er ai. 1987; Hart ettil., 1997: Knott and Wray. 2000; larvae from the identified larval groups are shown in Figure
KEK, unpubl. data). Regions between the tRNA genes were 1. This is the first study to identify field-collected cloning
also sequenced. These sequences were variable among the larvae.
asteroid taxa and could not be aligned. Since an assessment The largest group ofcloning larvae (group 1; 24 individ-
ofhomology ofthe inter-gene sequencescould not be made, uals) has sequences with high similarity to those from two
they were eliminated from the data set. Oreaster species, which group basally to the larvae. Both
The combined aligned data set was analyzed with multi- larval group 1 and the Oreaster clade are well supported
ple distance criteria using PAUP* ver. 4.0b4a (Swofford, (bootstrap percentages; 100 and 73 respectively), as is the
1998) to assess the degree ofsequence similarity. Sequence grouping of larval group 1 with the Oreaster clade (100%
differences were calculated first as uncorrected (p) distance, bootstrap support). Oreaster reticulatus is common in the
a measure of the number of aligned sequence positions tropical western Atlantic where the cloning larvae were
containing non-identical nucleotides divided by the total collected, whereas O. occidentalis is found in the eastern
number of positions compared. Genetic distance was then Pacific. The only other species of Oreaster in the Atlantic
calculated using modified methods that account for super- Ocean is O. clavatus, found in the eastern Atlantic from
imposed mutational events according to the Jukes-Cantor Cape Verde to the Gulf of Guinea (Clark and Downey,
and Kimura 2-parameter models of evolutionary change 1992). O. clavatns was not included in this analysis, and
(described in Swofford et ai. 1996). Genetic distances may be a good candidate species for the identity of larval
obtained from all methods were then used in neighbor- group 1. The genetic distance observed between the
joining analysis. Differences in trees obtained using differ- Oreaster species and larval group 1 is not large (range:
ent genetic distance calculations were assessed with the 0.06745 to0.10026: average: 0.08262) and is comparable to
Kishino and Hasegawa (1989) and Templeton (1983) non- other intra-genus distances (Table 3). However, some very
parametric tests in PAUP*. Stability ofclades in the result- closely related genera (within the same family) have genetic
ing trees was assessed by bootstrap analyses (1000 repli- distances as low as that seen between larval group 1 and the
cates; Felsenstein, 1985). Oreasterclade. Thus, it is also possible that the larvae could
belong to a species in a genus closely related to Oreaster. If
Results and Discussion so, the species identity oflarval group 1 must lie with avery
close relative of Oreaster. perhaps within its taxonomic
Neighbor-joining analysis using three methods of calcu- family, the Oreasteridae, or within another closely related
lating genetic distances resulted in only slightly different family, the Asteropseidae (Blake, 1987).
tree topologies. Analysis of uncorrected (/;) distances and Generic identification is more certain for a smaller group
Jukes-Cantor distances yielded identical tree topologies. of cloning larvae (group 2; 5 individuals) and a single
Analysis of Kimura 2-parameter distances yielded a longer cloning larva, both of which fall within a clade of Litidia
tree topology that was statistically different when tested species (96% bootstrap support). There are seven Liiidia
with the Kishino and Hasegawa test (P -- 0.013) and species in the tropical and subtropical western Atlantic
=
Templeton nonparametric test (P 0.013). However, be- Ocean (Clark and Downey. 1992). Our analysis includes
cause none of the relationships in clades including cloning twoofthese, as well as otherLnidia species from the Pacific
larvae were affected by the genetic distance calculation Ocean. The western Atlantic species not represented here
used, only the treegenerated using uncorrected (/>) distances are good candidates for the species identity of larval group
is shown and discussed (Fig. 2). 2 and the single cloning larva contained in this clade.
Analysis of sequence similarity identified four distinct Relationship ofthe five individuals in larval group 2 is well
groups of cloning larvae. Within these groups there was supported, in 100%- ofbootstrap replicates. The single clon-
very little sequence divergence (/> distances ranging from ing larva does not group with larval group 2, and instead is
0.00000 to 0.02589), indicating that each group most likely unresolved in a polytomy with the Pacific Lnidia species.
represents a single species (Table 3). However, neighbor- The grouping of this individual with the Pacific species is
joining analysis ofthe tRNA sequences did not place any of only moderately supported with bootstrap analyses (68%),
the 44 known adult asteroid species analyzed here within so its affinities tootherLnidia species are unclear. However,
the larval groups (Fig. 2). The field-collected larvae thus genetic distance between this individual and larval group 2
cannot be identified to species with the limited number of is high (averaging 0.08958). Since genetic distances within
sequences from i nown asteroid species available for com- larval group 2 are over 30 times lower (average: 0.00278),
parison. However, the close assemblage ofadults ofknown it is unlikely that the single cloning larva is also member of
taxa with the four larval groups identifies those genera or this group. Likewise, genetic distances between known Ln-
families with species capable oflarval cloning and indicates idia species and larval group 2 are also high (range: 0.07524
ASTEROIDS CAPABLE OF LARVAL CLONING 251
Op/iid/asterop/iidianus
larval group3, 2 individuals
larval group4, 12 individuals
I IJlU'kiamull/fora
~LLiinncckficiaagguiu/idliantmfgiiT.I
Linckiaguitdingi^
Luidiasenegalfnsis
Luidiaclathrata
Luidiaclal/iratalarva
"single cloning larva
Luidiaitiagel/anica |
Luidiafd/iii/ata
larval group 2, 5 individuals
Luidiaallernata
54 .J'.s-itsntirpttepcetcetenriardtuipi/-iiic/aattuuss
Pseudarchasterparelli
Ctenodiscuscrispatns
.-Islerinapei-finifera
PaUriellavii'ipara
Dermasteriasimbricata
larval group 1, 24 individuals
Oreasterreticiilatus
Oreasteroccidentalis
.-leant/tasterplanci
Afediasreraequa/is
' ffippasteriaspinosa
Diplasiasterproductus
Tosiaparya
95, Arc/tastert")'piEccushinaster/iindestus
-L .fft'nriciasp.
100, So/asterstimpsoni
Crossasterpapposus
Pisasteroc/iraceus
701 Stylasteriasfnrreri
Orlhasreriaskoe/i/eri
Kat/ihuiiaMercaliforniiy/.r
Pycnvpadiahelianf/widcs
Evasteriastroscbelli
Lfplasleriasliexactis
Urasreriaslincki
.isteriasforbesi
Brisingasterrobillardi
ffeliasterhelianthoides
Aft'xoderntaplat)'acanthus
Heiiriciaantillarnni
Pterastertesselatus
__ Hymenasterpellucidus
PlerasterJordani
Echinoidea
Figure 2. Phylogenetic tree resulting from neighbor-joining analysis of imairrected (/)) distances between
mitochondrial tRNA sequences of known asteroid species and field-collected cloning larvae. Field-collected
cloning larvae fall into four distinct groups and one single cloning larva (boxed), which are phylogenetically
widespread.Numbersoflarvaeineachlarvalgroupareindicated.Numbersatnodeswithinthetreearebootstrap
percentages from 1000 replicates. Larval tRNA sequences are accessioned in GenBank under numbers
AY249946-AY249978. GenBank accession numbers for known asteroid taxa include some published previ-
ously (Himeno ?f /.. 1987; Hart ft <//.. 1997: Knott and Wray. 2000) and AY245490-AY245506.
to 0.11438), similar to those seen between species of the tances determined in this and other studies are very low
same genus (Table 3). The sequence differences between (Table 3). Sequencesfromaknown larvaofL cluthrata and
larval group 2, the single cloning larva, and other Luidia an adult representative of this species from a different
species suggests that multiple species within Luidia are locality are identical and group together in neighbor-joining
capable oflarval cloning. The alternative, that there may be analysis with 100% bootstrap support (Tables 2 and 3).
genetic variation within species complicating our similarity The remaining two groups of cloning larvae (group 3
analyses, is not likely given that intra-species genetic dis- with 2 individuals and group 4 with 12 individuals) show
252 K. E. KNOTT ET AL.
Table 3
Genetic distances obsen'edbetween asteroid ta\a at difjerent ia.\nnomie levels: genetic distance reportedis calculatedfrom tRNA genes I*) or the
COIgene I I
Taxonomic Level
ASTEROIDS CAPABLE OF LARVAL CLONING 253
Table 4
Generic distances benveen the 12 individuals in larvalgroup 4 andclosely relatedtaxa in the Ophidiasteridae, calculatedusing methodsfor
uncorrecled(p) distance
TAXA
3. 4. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
1. larva 1
2. larva 2
3. larva 3
4. larva4
5. larva 5
6. larva 6
7. larva 7
254 K. E. KNOTT ET AL
Wray, 1996; Hurt, 2000). Despite being only recently con- Chia, F. S., and C. \V. Walker. 1991. Echinodermata: Asteroidea. Pp.
firmed in asteroids (Bosch el /.. 1989; Jaeckle 1994), larval 301-353 in Reproduction ofMarine Invertebrates, Vol VI: Echino-
cloning may have an ancient evolutionary origin. eddcsr.msBoaxnwdoLoodphPorpehsosr,aPtaecsi,ficA.GrC.ovGei,esCeA,.J. S. Pearse,andV. B. Pearse,
The larval groups, or species, discerned here consist of Clark, A. M., and M. E. Downey. 1992. Starfishes of the Atlantic.
multiple cloning larvae collected from different localities. Chapman and Hall. London.
Widespread occurrence of cloning larvae may not be sur- Domanski, P. A. 1984. Giant larvae: prolonged planktonic larval phase
prising, since planktonic larvae of asteroids can disperse in the asteroid Luidia sarsi. Mar. Biol. 80: 189-195.
great distances. In addition, the adults of many asteroid Felsenstein, J. 1985. Confidence limits on phylogenies: an approach
species have broad geographic ranges that in some cases GenuesiCnogdtehse bCooortpsotrraapt.ioEnv.olu1t9i9o5n.39S:ci7/8u3c-nc7l9i1e.r 3.0. Gene Codes Corpo-
extend beyond the area sampled here. The identification of ration. Ann Arbor. Ml.
different modes of larval cloning within one species (larval Hart, M. W. 200(1. Phylogenelic analyses of mode of larval develop-
group 1) is a bit more surprising, despite observations of ment. Seniin. CellDev. Biol. 11: 411-418.
multiple modes of cloning in laboratory-cultured species Hart, M. W., M. Byrne, and M. J. Smith. 1997. Molecular phyloge-
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different developmental regimes for the resulting clones 230.
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Many thanks go to Bill Allison, Richard Emlet. Igor Kitazawa,C.,and M. Komatsu.2001. Larvaldevelopmentandasexual
Gorchakov, Dan Janies, Sue Lisin. Christopher Lowe, Chris developmentoftheseastar,Disti>Uisterinsni/ion(Doderlcm). P. 177 in
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by a National Geographic Society grant 6267-98 to E. J. Knott, K. E., and G. A. Wray. 2000. Controversy and consensus in
Balser and W. B. Jaeckle. We also thank the Smithsonian asteroidsystematics: newinsightstoordinal andfamilial relationships.
Am. loot. 40: 382-392.
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