Developmental morphology of the cyprinid fish, Candidia barbatus

 Embryonic, larval, and juvenile development of a Taiwanese cyprinid fish, Candidia barbatus, is described from laboratory-reared specimens. The eggs, measuring 1.8–2.1 mm in diameter, were demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk and no oil globule. Hatching occurred 56–69 h after fertilization, the newly hatched larvae measuring 4.9–5.3 mm in body length (BL) with 25–26 + 13–14 = 39–40 myomeres. The yolk was completely absorbed at 7.6 mm BL. Notochord flexion was initiated at 6.8 mm BL and finished at 7.6 mm BL. Aggregate numbers of all fin rays were completed at 12 mm BL. Barbels on the upper jaw appeared near the corner of the mouth at 17 mm BL. Eggs of the species closely resembled those of its related cyprinid genera, Opsariichthys and Zacco. Larvae and juveniles of C. barbatus were similar to those of O. uncirostris subspp., Z. platypus, and Z. pachycephalus, but differed from the latter in the process of disappearance of the adipose finfold (postflexion larval stage), barbels on upper jaw (juvenile stage), and pigmentation on the lateral body surface (postflexion larval and juvenile stages). Although C. barbatus also differed from the Z. temminckii species' group [Z. temminckii and Zacco sp. (sensu Hosoya, 2002)] in having barbels, larvae and juveniles of the former showed more similarity to the latter species group than to O. uncirostris subspp., Z. platypus, and Z. pachycephalus, from the aspect of head and body pigmentation.     

The taxonomy and phylogeny of the cyprinid genus Opsariichthys Bleeker (Teleostei: Cyprinidae) from Taiwan, with description of a new species

The morphological and mitochondrial genetic differentiation in the cyprinid genus, Opsariichthys Bleeker (Nederlandsch Tijdschrift voor de Dierkunde 1:187–218, 1863) have been surveyed in Taiwan. Among them, there are three valid species can be recognized in Taiwan including Opsariichthys pachycephalus Günther (1868) distributed in northern and western Taiwan, Opsariichthys evolans (Jordan and Evermann Proc US Nat Mus 25:315–368, 1902) in northern Taiwan and an unnamed species from southern Taiwan described herein as Opsariichthys kaopingensis Chen and Wu, new species which can be well distinguished from the related O. pachycaphalus by their body proportions, scale counts, and specific coloration patterns. We utilized mitochondrial complete D-loop sequence data to infer phylogenetic relationships within a subset of related genera of opsariichthines, and to examine evidence for genetic differentiation in these two sibling species formerly assigned to “Zacco” pachycephalus and their genetic relationship with other congeneric species around nearby regions. The clade of O. pachycephalus and O. kaopingensis in genetically were recovered as more closely related to Opsariichthys uncirostris (Temminck and Schlegel 1846) species complex including both O. uncirostris and O. bidens Günther (1868) from Japan and mainland China than to typical Zacco from Japan. This molecular phylogenetic insight strongly supports the assignment for both so-called “Zacco” pachycephalus and this new species described herein as the typical monophyletic members of Opsariichthys and the type species of Zacco as Zacco platypus (Temminck and Schlegel 1846) from Japan is sister clade for all species groups in Opsariichthys. Opsariichthys pachycephalus and O. kaopingensis were strongly differentiated by large mitogenetic distances and phylogenetic support from distance and discrete method and Bayesian inference based on complete mtDNA D-loop sequences, with an average mitogenetic divergence of 3.3%, which may suggest that the separation of the two species happened much earlier than the last glacial period. Opsariichthys evolans seems to share the close genetic relationship with O. acutipinnis (Bleeker Nederlandsch Tijdschrift voor de Dierkunde 1:187–218, 1863) from the Yangtsi River basin.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Varicorhinus alticorpus</Emphasis> (Cyprinidae)

Varicorhinus alticorpus (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the southern and eastern regions of Taiwan. We described the development of nine polymorphic microsatellite loci in V. alticorpus for genetic studies. These new markers were tested in 20 individuals of the rare species. The number of alleles ranged from 4 to 13. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.692 to 0.892 (averaged at 0.821) and from 0.000 to 0.350 (averaged at 0.088), respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. This suite of highly polymorphic microsatellites provides the first chance to undertake a conservation program for this species in Taiwan. It is essential to evaluate its genetic diversity and the fine-scale population structure. Tzen-Yuh Chiang, Teh-Wang Lee, and Feng-Jiau Lin contributed equally to this work.     

Isolation of polymorphic microsatellite loci in the clear lake gnat and two other phantom midge species of the genus Chaoborus (Diptera: Chaoboridae)

Chaoborus is of great interest to many freshwater ecologists. The adults can become pests in certain areas in North America and the larvae are an important food source for fish. In this preliminary study, we identified variable microsatellite loci in three species: Chaoborus astictopus (H_E = 0.52–0.76), Chaoborus americanus (H_E = 0.46–0.80) and Chaoborus punctipennis (H_E = 0.66–0.81). Using a biotin/streptavidin capture technique of repetitive sequences in a 96‐well format, we obtained microsatellite‐enriched genomic libraries for all three species and identified six polymorphic microsatellite markers for each species. None of the primers did yield a polymerase chain reaction fragment in a cross‐species test.     

Mitochondrial phylogeny reveals the artificial introduction of the pale chub Zacco platypus (Cyprinidae) in Taiwan

The presence of the pale chub Zacco platypus (Japanese name, Oikawa) in Taiwan has been suggested to be a result of its inadvertent introduction from Lake Biwa in Japan in the 1980s in conjunction with the Japanese Ayu, Plecoglossus altivelis altivelis, which was released several times into the Tamsui River to restock the extinct Ayu population of Taiwan. However, it is also possible that Z. platypus is native to Taiwan and has not been previously described for reasons of its narrow range. Knowledge of the colonizing history of Z. platypus is of considerable importance because it provides insight into the evolutionary process and, hence, impacts management decisions regarding this species in Taiwan. A portion of the mitochondrial D-loop was sequenced for 77 specimens from five populations of Z. platypus from Japan and Taiwan. A total of 22 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.20% to 2.82%. Haplotype diversity was high in all populations examined, with a range from 0.718 in the Tagiri River population to 0.909 in the Lake Biwa population. Phylogenetic and statistical parsimony analyses of the molecular data revealed a close genetic relationship between Taiwanese and Japanese Z. platypus and supported the previous report that the Taiwanese Z. platypus originated in Lake Biwa in Japan.     

Isolation and characterization of microsatellite loci in Shortia rotundifolia (Diapensiaceae), an endangered relict plant on the Ryukyu Islands and Taiwan

Ten microsatellite loci were isolated and characterized for Shortia rotundifolia, a relict temperate plant on the subtropical Ryukyu Islands and Taiwan. The number of alleles ranged from 2 to 21. The expected (H _E) and observed (H _O) heterozygosities were 0.027–0.877 and 0.027–0.622, respectively, from 74 individuals on Iriomotejima Island of Ryukyus. One locus exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). These primers amplifying microsatellites in this species may provide a useful tool for population genetics to establish conservation strategy.     

Limited cross‐species microsatellite amplification and the isolation and characterization of new microsatellite markers for the greater stick‐nest rat (Leporillus conditor)

The greater stick‐nest rat (Leporillus conditor) is a conilurid rodent whose recent history provides an opportunity to examine genetic changes in reintroduced populations. We trialled 63 known microsatellite primers from Rattus rattus and Mus musculus in L. conditor. Three primer pairs produced polymorphic loci (number of alleles = 2–3, mean, H_E = 0.42). Subsequently, we isolated and characterized 12 novel polymorphic microsatellites (mean number of alleles = 5–16, mean H_E = 0.76) from L. conditor from genomic libraries for use in population genetic studies.     

Isolation and characterization of 23 polymorphic microsatellite markers for diversity and stock analysis in tiger shrimp (Penaeus monodon)

The tiger shrimp (Penaeus monodon) is an important marine crustacean in terms of biological diversity and aquaculture resource. The shrimp is widespread across the Indo‐Pacific region and shows apparent genetic differentiation among geographical populations. It is common practice to transport female brooders between different countries to seed the shrimp farms, posing potential problems of unwanted population admixture. We developed 23 polymorphic microsatellites for P. monodon (average H_E = 0.936) and these microsatellites were applicable for studying population differentiation, identifying valid stocks and tagging nonindigenous farmed shrimps.     

Noninvasive individual and species identification of jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) in Belize,Central America using cross‐species microsatellites and faecal DNA

There is a great need to develop efficient, noninvasive genetic sampling methods to study wild populations of multiple, co‐occurring, threatened felids. This is especially important for molecular scatology studies occurring in challenging tropical environments where DNA degrades quickly and the quality of faecal samples varies greatly. We optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) and assessed their utility for cross‐species amplification. Additionally, we tested their reliability for species and individual identification using DNA from faeces of wild felids detected by a scat detector dog across Belize in Central America. All microsatellite loci were successfully amplified in the three target species, were polymorphic with average expected heterozygosities of H_E = 0.60 ± 0.18 (SD) for jaguars, H_E = 0.65 ± 0.21 (SD) for pumas and H_E = 0.70 ± 0.13 (SD) for ocelots and had an overall PCR amplification success of 61%. We used this nuclear DNA primer set to successfully identify species and individuals from 49% of 1053 field‐collected scat samples. This set of optimized microsatellite multiplexes represents a powerful tool for future efforts to conduct noninvasive studies on multiple, wild Neotropical felids.     

Genetic differences between continuous and disjunct populations: some insights from sal (<Emphasis Type="Italic">Shorea robusta</Emphasis> Roxb.) in Nepal

Sal (Shorea robusta Gaertn., Dipterocarpaceae) is a wind-pollinated tropical tree species found in southern Asia. We investigated the genetic diversity and structure at four microsatellites of 15 populations comprising continuous-peripheral and disjunct-peripheral populations in Nepal. Estimates of genetic diversity (N _A = 8.98, H _O = 0.62, H _E = 0.69) were similar when compared with those of other tropical tree species. A higher level of genetic diversity was observed in continuous-peripheral populations (N _A = 9.61, H _O = 0.67, H _E = 0.72) as compared to disjunct-peripheral (N _A = 8.04, H _O = 0.55, H _E = 0.64). Population differentiation was higher among disjunct-peripheral populations (F _ST = 0.043) than among continuous peripherals (F _ST = 0.012). There was a significant association between gene flow distances and genetic differentiation (r ² = 0.128, P ≤ 0.007). No spatial arrangement of populations according to their geographical locations was found. Based on observed genetic diversity protection of some populations in continuous-peripheral range are suggested for the sustainable conservation of genetic resources of the species while protection of some disjunct-peripheral populations are also recommended for conserving rare alleles.     

The reproductive biology of an endemic cyprinid,Zacco pachycephalus,in Taiwan

Synopsis Reproductive parameters including body weight-length relationship, condition factor, gonadosomatic index (GSI) and fecundity of an endemic cyprinid,Zacco pachycephalus, were studied from October 1991 to January 1993. Although the condition factor was similar between both sexes, GSI values of female (3.72 to 14.24%) were significantly larger than that of males (0.21 to 2.23%) during the major breeding season. The number of yolky eggs per fish ranged from 459 to 2059. Ovum size distribution, annual GSI value, and recruitment of juvenile fish indicated thatZ. pachycephalus conducted two major spawning activities in spring (February to April) and summer (June to August). The larger GSI value and number of yolky eggs in spring compared to those in summer implies thatZ. pachycephalus has evolved a reproductive style by investing more energy in spring to avoid the damage of summer freshet on its recruitment success. The mating behavior ofZ. pachycephalus is similar to that of otherZacco fishes.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Isolation and characterization of polymorphic microsatellite DNA markers in topmouth gudgeon,Pseudorasbora (Teleostei: Cyprinidae)

Due to accidental introductions, the distribution of the east Asian topmouth gudgeon, Pseudorasbora parva, has rapidly expanded over recent years, thereby threatening the endangered Japanese species, P. pumila pumila. Sixteen microsatellite loci were isolated and characterized for P. parva, 14 being highly polymorphic (mean H_E = 0.58, mean number of alleles = 4.4). Successful characterization of 10 of these loci was also achieved for P. pumila pumila. These markers should be useful in future investigation of colonization of P. parva and the development of conservation strategies for P. pumila pumila.     

Comparative phylogeography of Zacco platypus and Opsariichthys bidens (Teleostei, Cyprinidae) in China based on cytochrome b sequences

We compared the phylogenetic relationships and the population structure of the sympatric cyprinids Opsariichthys bidens and Zacco platypus across the Yangzte, Pearl and Hai He drainages in Mainland China. We used the mitochondrial cytochrome b gene to evaluate their phylogeographical structure and to discuss further systematic implications. We sequenced 71 new individuals of Z. platypus to add to a previous study and to construct a geographically comparable data set with O. bidens (Mol. Phylogenet. Evol. 31, 2004, 192; Mol. Phylogenet. Evol. 37, 2005, 920). Phylogeographic analysis of Z. platypus identified two additional mtDNA lineages (Zacco E and F) in addition to the previously recovered lineages (Zacco A–D). The geographical range of the Zacco A was extended across the Pearl drainage, broadening considerably the previous distribution that was circumscribed to the Yangtze drainage. Our results show high molecular divergence between all Zacco A–F mtDNA lineages (mean TrN + I + G distance 17.7%, range 4.4–30.8%), and the population analyses showed that most of the genetic variation was found among major river tributaries (90.2%). Values of θ_ST higher than 0.97 corroborated this interruption of gene flow between Zacco A–F mtDNA lineages. Nested clade analysis results were also concordant with the phylogenetic relationships recovered. We found similar phylogeographic patterns in Z. platypus and O. bidens: they exhibited two widespread and common lineages (Zacco A and Opsariichthys 1) ranging across the Yangtze and Pearl drainages, and both taxa had Northern lineages (Zacco F and Opsariichthys 5) clearly differentiated. Similar phylogeographic patterns suggest that common historical factors have shaped current Zacco and Opsariichthys diversity. High genetic distances and θ_ST values suggested that differentiation of Zacco A–F mtDNA lineages and Opsariichthys 1–5 was not recent and was probably favoured by old geological events. However, population parameters and low genetic differentiation inside Zacco A and Opsariichthys 1 suggest a recent gene flow across river drainages. Mitochondrial‐based phylogenies for the combined data sets of Zacco A–F and Opsariichthys 1–5 mtDNA lineages always recovered both taxa as paraphyletic, highlighting the notion that their current systematics need a revision.     

Polymorphic microsatellites in two different species of the genus Zoogoneticus Meek, 1902 (Goodeidae,Actynopterygii)

This report describes the characterization of 13 microsatellites in Zoogoneticus tequila, a goodeid fish that is extinct in nature and exists only in captivity. We also present a preliminary survey of the variability of nine of these microsatellites in both Z. tequila and the congeneric wild species Z. quitzeoensis. Variability was found to be low in Z. tequila (three polymorphic loci with two alleles each), and highest in Z. quitzeoensis (five polymorphic loci with a mean of 4.6 alleles). These genetic markers have applications in the study and management of both species.     

Tandem repeat markers for population genetic studies of the protected California tiger salamander (<Emphasis Type="Italic">Ambystoma californiense</Emphasis>)

Habitat loss is the single greatest threat to persistence of the critically threatened California tiger salamander (Ambystoma californiense). To aid management plans that designate critical habitat for this species, I developed and characterized 21 tetranucleotide microsatellite markers using two native populations in Santa Barbara and Alameda Counties. Allelic variation and average heterozygosities were lower in the endangered Santa Barbara population (allele range 1–4, mean 2.4; H _O = 0.308 H _E = 0.288) compared with the threatened Alameda population (allele range 2–10, mean 6.7; H _O = 0.712, H _E = 0.722). In-depth population studies using these markers will provide vital information for plans to assign critical habitat that optimize gene flow among breeding populations, as well as for identifying non-native hybrid genotypes that threaten native A. californiense stocks. Beyond the conservation goals for A. californiense, the close phylogenetic relationships within the tiger salamander complex also suggest a broad utility for population studies using these markers.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

<Emphasis Type="Italic">Zacco sieboldii</Emphasis>, a species distinct from <Emphasis Type="Italic">Zacco temminckii</Emphasis> (Cyprinidae)

 Two biological types of Japanese dark chub, so-called types A and B of Zacco temminckii, were taxonomically inspected. A comparison of types A and B with the lectotypes of Leuciscus sieboldii and L. temminckii in Siebold's collection revealed that type A is identical to L. sieboldii, whereas type B matches L. temminckii. Hence, Zacco sieboldii and Z. temminckii were redescribed on the basis of the lectotype and additional specimens from Japan. Zacco sieboldii is distinguishable from Z. temminckii by having a narrower band on the anterior portion of both body sides, nine branched rays of anal fin (10 in Z. temminckii), lateral line scales not less than 53 (not more than 52 in Z. temminckii), and scales above lateral line not less than 13 (not more than 11 in Z. temmincki). A key to the species of Japanese Zacco is also provided. Received: August 15, 1999 / Revised: July 25, 2002 / Accepted: August 19, 2002 Acknowledgments We express our cordial thanks to all the following investigators: Dr. M.J.P. Van Oijen (RMNH) for the specimen loan of von Siebold's collection; Professor San-Rin Jeon, Sang-Myung University (formerly, Seoul) for providing several papers on Korean Zacco congeners; Dr. Harumi Sakai, National Fisheries University; Dr. Tetsuo Furukawa-Tanaka, Museum of Nature and Human Activities, Hyogo; Dr. Seishi Kimura, Mie University; Dr. Akihisa Iwata, Kyoto University; Dr. Osamu Katano, National Research Institute of Fisheries Sciences; Mr. Kazuo Hoshino, Oita Marine Palace; and Dr. Kouichi Kawamura, National Research Institute of Aquaculture, for the valuable comments and information on the ecological aspects of both species of the Japanese dark chub. Correspondence to:Kazumi Hosoya     

Isolation and characterization of microsatellite loci in the Cape fynbos heath Erica coccinea (Ericaceae)

Enriched genomic libraries were used to isolate and characterize dinucleotide microsatellite loci in Erica coccinea, a South African Cape fynbos heath species with distinct resprouter and seeder populations. Microsatellites were required to investigate the effect of the contrasting demographic pattern driven by these two post-fire responses in the population genetic structure of seeder and resprouter forms within this species. Eight microsatellite loci were characterised and amplified a total of 106 alleles in 2 samples each of 30 individuals from 1 resprouter and 1 seeder population. Mean allele numbers were 7.88 and 11.0 for the resprouter and seeder population, respectively. Both populations showed similar average observed and expected heterozygosity levels, H _O(resprouter) = 0.683, H _O(seeder) = 0.696; H _E(resprouter) = 0.726, H _E(seeder) = 0.756, and average positive inbreeding coefficients F _IS(resprouter) = 0.058, F _IS(seeder) = 0.080. This set of microsatellite loci will be used to conduct a population genetic survey of seeder and resprouter populations throughout the range of the species. Cross-species transferability was also assayed in four other South African and four European species of the genus Erica, supporting their potential use for population genetic analyses.     

Isolation and characterization of 12 microsatellite loci from <Emphasis Type="Italic">Suzukia shikikunensis</Emphasis> (Lamiaceae), a genus endemic to Taiwan and Ryukyus

Suzukia shikikunensis Kudo is an endemic plant in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, we developed 12 microsatellite primer pairs for genetic study. These markers were screened for 24 samples collected from wild populations distributed in Taiwan, and for a sister species S. luchuensis collected from Yonaguni and Lutao Islands. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.65–0.922 and 0–0.625, respectively. All loci were significantly deviated from Hardy–Weinberg equilibrium due to the heterozygote deficiency. These primers amplifying microsatellites in the two species may provide a useful tool for population genetics to establish conservation strategy.