Phylogeny of the slow lorises (genusNycticebus): An approach using mitochondrial DNA restriction enzyme analysis

Mitochondrial DNA polymorphisms in 15 specimens of three species of slow lorises-Nycticebus coucang, N. intermedius, andN. pygmaeus-were analyzed in order to study the evolutionary relationships among the species. Eight restriction types were observed in the samples. Phylogenetic trees constructed on the basis of genetic distances showed that the slow lorises sort into two clusters: four types ofN. coucang and three types ofN. intermedius plus one type ofN. pygmaeus. Our results suggest that there are two valid species in the genusNycticebus-N. coucang, andN. pygmaeus-and thatN. intermedius should be included withinN. pygmaeus. Divergence between the two species may have begun 2.7 Ma (million years ago). Evolution of gross morphology, chromosomes, and mitochondrial DNA in the slow lorises appears to be concordant.     

Studies on the chromosomes of genusNycticebus

The karyotypes of three species (N. coucang, N. intermedius, andN. pygmaeus) of genusNycticebus, collected from the southern Yunnan of China, have been studied. All individuals from three species possess 2n=50 chromosomes, and all chromosomes in their complement are biarm chromosomes. The karyotype of slow loris (N. coucang) is characterized by having a secondary constriction and Ag-NORs in the short arms of pair No. 1. The G-banding patterns of three species are very similar. Three species are found to have multiple Ag-NORs. InN. coucang, NORs were observed on five pairs (Nos. 1, 6, 9, 15, and 23) and inN. intermedius andN. pygmaeus, NORs were found on four pairs (Nos. 6, 9, 15, and 20). This finding indicates that slow lorises, as primitive primates, also have multiple NOR-bearing chromosomes. Finally, the classification of genusNycticebus by karyotype analysis is discussed, and our results suggest that there are at least two valid species, namely:N. coucang andN. pygmaeus.     

Taxonomy of theAlouatta seniculus group: Biochemical and chromosome data

Wild populations ofAlouatta belzebul andA. seniculus from Brazil were surveyed in relation to 20 protein loci. Estimates of genetic variability demonstrated thatA. seniculus presents the highest level of heterozygosity among several New World monkey species studied for the same loci. Additional information from DNA and chromosomes suggest thatA. seniculus andA. belzebul are not the closest species in theHershkovitz’sAlouatta seniculus group.     

Genetic relationships between European and Siberian larch,Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species?

Thirteen populations ofLarix decidua subsp.decidua and subsp.polonica, and three populations ofL. sibirica were investigated by starch-gel electrophoresis. In the populations assayed 61 alleles at 17 loci were revealed. The allozyme data support the earlier observations about close relationships between these two larch species. Nei's genetic distances betweenL. decidua andL. sibirica were relatively small (D = 0.057), however, almost five times larger, on average, than those between populations of the same species. Results obtained in this study disagree withBobrov's hypothesis about the hybrid origin of the Polish larch and suggest a direct origin from the European larch.     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

Protein variation,taxonomy and differentiation in five species of marmosets (genusCallithrix Erxleben, 1777)

The electrophoretic patterns of 15 protein systems codified for 20 genetic loci were investigated using horizontal electrophoresis. A total of 150 blood samples, from five species of the genusCallithrix were analyzed. Polymorphic variation was observed in 10 out 20 loci analyzed. The genotypic distributions are in Hardy-Weinberg equilibrium. The average heterozygosity (H) varied from 1% to 5%, similar to those observed for other Neotropical primates. The genetic distance coefficients revealed a phylogenetic separation of these species into two groups: (1) “argentata” (C. humeralifer andC. emiliae); (2) “jacchus” (C. jacchus, C. penicillata, andC. geoffroyi). This arrangement is according to the taxonomic arrangement proposed byHershkovitz (1977),de Vivo (1988), andMittermeier et al. (1988). The results in each group are compatible with the subspecies values recorded for the Platyrrhini. These values showed that:C. humeralifer andC. emiliae are subspecies ofC. argentata;C. jacchus, C. penicillata, andC. geoffroyi are subspecies ofC. jacchus. These results also suggest thatC. j. geoffroyi is the “jacchus” group taxon, most similar genetically to the “argentata” group.     

A cline of allozyme variation inAbies mariesii

Genetic variation at 22 allozyme loci was examined for 1,003 trees from 11 isolated natural populations ofAbies mariesii covering all except the southernmost region of its geographic range. Genetic diversity within species (H _es=0.063) was low compared to many other long-lived woody species. Most of the genetic variation is found within populations (G _ST=0.144) despite their isolated distribution. Genetic distance between populations was positively correlated with geographic distance. Genetic diversity within populations was generally low (meanH _ep=0.054), but varied across populations in a clinal fashion such that genetic variation decreased with increasing latitude. These genetic characteristics may reflect the distribution history of this species.     

Genetic architecture in bur oak,Quercus macrocarpa (Fagaceae), inferred by means of spatial autocorrelation analysis

Allozyme variation at seven putative polymorphic gene loci (Fle, Idh, Mdh-2, Mdh-3, 6 Pgd-2, Pgi-2, Pgm) was quantified in an isolatedQuercus macrocarpa population in northwestern Ontario, Canada. The bur oaks studied were slightly less genetically variable (H _e = 0.196,A = 3.43) than theQuercus subg.Lepidobalanus average. To assess the genetic differentiation within the topodeme,Moran's spatial autocorrelation coefficients were used for the nearest-neighbor map and correlograms. Results indicate that most alleles were randomly distributed in space. However, significant genetic patches were detected for twoFle-alleles. Patch width was inferred to be less than 10m. This spatial substructure may be due to limited gene flow and/or demographic factors. Spatially varying selection can be probably excluded as a spatial evolutionary force, since site conditions were very homogeneous.     

Cranial allometry and geographic variation in slow lorises (Nycticebus)

A series of 20 craniodental measurements was obtained for two sister taxa: Nycticebus coucang (common slow loris) and N. pygmaeus (pygmy slow loris). Multivariate analysis of variance was performed with adult data to describe patterns of subspecific and specific variation in this genus. The geometric mean of adult cranial dimensions was compared to field data on latitudinal coordinates for available specimens to investigate if size variation in Nycticebus is clinal in nature. Ontogenetic series for larger-bodied N. coucang and smaller-bodied N. pygmaeus were compared to test the hypothesis that species and subspecific variation in skull form results from the differential extension of common patterns of relative growth. A MANOVA provides independent support of Groves's [pp. 44–53 in Proceedings of the Third International Congress on Primatology, Vol. 1 (Basel: S. Karger), 1971] classification of Nycticebus into two species, with four subspecies in the common slow loris and one form of the pygmy slow loris. Within N. coucang, cranial proportions for all four subspecies are ontogenetically scaled, and size differentiation is mainly clinal (Bergmann's Rule). N. c. bengalensis represents the most northerly disposed and the largest form. N. c. javanicus represents the next-largest form and is located in a southerly direction the next-farthest away from the equator. N. c. coucang and N. c. menagensis are both equatorial; however, the latter subspecies is the smallest. A genetic basis for some of the taxonomic variation between N. c. coucang and N. c. menagensis is supported by such nonclinal variation in body size. Variation in the presence/absence of I² is not size-related but rather tracks geographic proximity and isolating factors which predate the most recent inundation of the Sunda Shelf. Although they inhabit a nonequatorial environment, pygmy slow lorises are the smallest of all Nycticebus. As N. pygmaeus is sympatric with N. c. bengalensis, the largest slow loris, it appears that the evolution of its smaller body size represents a case of character displacement. Unlike N. coucang, skull size becomes significantly smaller in more northern N. pygmaeus. This may also reflect character displacement between sympatric sister taxa underlain by a cline-dependent ecological factor which is marked in more northerly latitudes. On the other hand, the negative correlation between body size and latitude in N. pygmaeus could be due to the influence of nonprimate fauna, such as predators, which themselves evince a similar clinal pattern. Analyses of relative growth indicate that skull proportions in the two species of Nycticebus are ontogenetically scaled in two-thirds of the cases. All but one of the seven comparisons (interorbital breadth) which do not indicate ontogenetic scaling represent part of the masticatory complex. This likely reflects a reorganization of N. pygmaeus maxillomandibular proportions linked to smaller size and changes in diet. Am. J. Primatol. 45:225–243, 1998. © 1998 Wiley-Liss, Inc.     

Genetic differentiation of theErigeron species (Asteraceae) in the Alps: A case of unusual allozymic uniformity

Allozyme variation was studied in all nine diploidErigeron species known from the Alps:E. alpinus, E. neglectus, E. polymorphus, E. candidus, E. uniflorus, E. atticus, E. gaudinii, E. acer, andE. angulosus. A total of 248 individuals from 24 natural populations was investigated using starch gel electrophoresis. Seven enzymes and 13 loci were assessed. Genetic variation within populations was low with the proportion of polymorphic loci ranging from 0.0–0.385, and average number of alleles per polymorphic locus from 2.0–2.5. In general, 70–100% of the genetic variation was attributed to between population differences. Mean genetic identities for pair-wise comparisons of populations averaged 0.893 within species, and 0.890 among species. Interspecific genetic variation of populations usually did not exceed intraspecific variation. It was concluded that theErigeron species from the Alps may have arisen by recent speciation probably during the epoches of glaciation. Morphological and ecological differences between species seem to be based on few gene loci.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

The genetic variability of the red king crab, <Emphasis Type="Italic">Paralithodes camtschatica</Emphasis> (Tilesius, 1815) (Anomura,Lithodidae) introduced into the Barents Sea compared with samples from the Bering Sea and Kamchatka region using eleven microsatellite loci

The intentional introduction of red king crab, Paralithodes camtschatica (Tilesius, 1815) in the Barents Sea represent one of a few successful cases and one that now supports a commercial fishery. Introductions of alien species into new environments are often associated with genetic bottlenecks, which cause a reduction in the genetic variation, and this could be important for the spreading potential of the species in the Atlantic Ocean. Red king crab samples collected in the Varangerfjord located on the Barents Sea (northern Norway) were compared with reference crab samples collected from the Bering Sea and Kamchatka regions in the Pacific Ocean. All samples were screened for eleven microsatellite loci, based on the development of species-specific primers. The observed number of alleles per locus was similar, and no reduction in genetic variation, including gene diversity and allelic richness, was detected between the Varangerfjord sample and the reference sample from Okhotsk Sea near Kamchatka, indicating no genetic bottlenecking at least for the microsatellite loci investigated. The same results were found in comparison with the sample from Bering Sea. The level of genetic differentiation among the samples, measured as overall F _ST across all loci, was relatively low (0.0238) with a range of 0.0035–0.1000 for the various loci investigated. The largest pairwise F _ST values were found between the Bering Sea and Varangerfjord/Barents Sea samples, with a value of 0.0194 across all loci tested. The lowest value (0.0101) was found between the Varangerfjord and Kamchatka samples. Genetic differentiation based on exact tests on allele frequencies revealed highly significant differences between all pairwise comparisons. The high level of genetic variation found in the Varangerfjord/Barents Sea sample could be of significance with respect to further spreading of the species to other regions in the North Atlantic Ocean.     

Electrophoretic analysis of the phylogeny of the self-pollinating populations ofClarkia xantiana

Electrophoretic variation in isozymes coded by 40 loci was examined in two self-pollinating populations, one with white and the other with pink flowering plants, and four outcrossing populations ofClarkia xantiana (Onagraceae) native to California. The study was carried out to test theMoore & Lewis (1965) hypothesis that the pink selfer originated from the sympatric outcrossing population and then gave rise to the white selfer. The hypothesis could be rejected if one or the other selfer was more similar genetically to an allopatric population than to the sympatric one. Both selfers were monomorphic at all loci whereas the outcrossing populations were polymorphic at nearly half of them. The two selfers had the same genes at 32 loci but had different ones at eight loci. The pink selfer was not more similar to the allopatric populations than to the sympatric one, consistent with theMoore & Lewis model. The evidence also supported their proposal that the white selfer originated from the pink one and not independently. The electrophoretic evidence was valuable because it permitted qualitative comparisons (presence versus absence of particular alleles) between the selfer and the several outcrossing populations; such analysis was not previously possible because the latter populations are morphologically and cytologically similar.     

Genetic differentiation without concordant morphological divergence in the thallose liverwortConocephalum conicum

Allozyme variation was examined at 33 enzyme loci for 96 populations from throughout the geographical range ofConocephalum conicum (L.)Dum. (Marchantiales, Hepaticae). Variation was partitioned into five discrete groups, suggesting that this morphologically defined species is genetically heterogeneous. The degree of differentiation among these groups, as measured by genetic distance, is as large as is commonly reported between different vascular plant species, and much larger than that between conspecific populations. In Europe, two of these genetically distinct groups (S and L) occur sympatrically, but apparently do not interbreed. Geographical ranges of the other three groups (A, C, and J) are probably allopatric with the exception that the range of S, the most genetically divergent group, overlaps with group A in N. America. It is suggested that these five natural assemblages constitute different sibling species.     

Evolution and systematic relationships in theTriticeae (Poaceae)

The evolution and taxonomic relationships in theTriticeae are discussed with the view to highlight aspects of this agronomically important group of plants, which may be of interest to molecular biology. Some of these aspects are addressed in more detail in adjoining papers in which specific genomic loci have been examined at the DNA sequence or isozyme level. Aspects discussed include the systematics and geographic distribution of theTriticeae species, isozyme and chromosome pairing studies on some of the species as well as more recent developments in DNA analyses. A survey of the systematics of theTriticeae indicated that the genomic system ofLöve is probably the most useful starting point for interpreting molecular data even though the system has many problems from a taxonomic point of view. The geographical distribution ofTriticeae species, using both published and unpublished data, suggested that information of this type taken together with the theory of continental drift provides a broad time-span for considering data from DNA sequence studies. The significance, and modes of analyses, of isozyme studies were assessed because they often provide valuable characters in determining relationships between species. The main character underlyingLöve's andDewey's analyses of theTriticeae, namely chromosome pairing, is discussed with particular reference to isozyme studies to show that in some cases, such as species ofHordeum sensu lato, consistent relationships are obtained. Finally, new developments in understanding chromosome structure are considered in relation to the above variables in the taxonomy and evolution of theTriticeae.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Genetic Diversity of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Revealed by PCR-SSCP of Polymorphic EST-Derived Markers

A total of 90 ESTs from normal and 157 from subtractive ovarian cDNA libraries of the giant tiger shrimp (Penaeus monodon) were sequenced. SSCP analysis of disulfide isomerase (DSl), zinc finger protein (ZFP), PMO920, and PMT1700 was carried out for population genetic studies of P. monodon in Thai waters. The number of codominant alleles per locus for overall samples was 6 for PMO920, 5 for PMT1700, and 12 for ZFP, and there were 19 dominant alleles for DSI. The observed heterozygosity of each geographic sample was 0.3043–0.5128 for PMO920, 0.3462–0.4643 for PMT1700, and 0.5000–0.8108 for ZFP. Linkage disequilibrium analysis indicated that genotypes of these loci segregate randomly (P > 0.05). Low genetic distance was found between pairs of geographic samples (0.0077–0.0178). The neighbor-joining tree constructed from the average genetic distance of overall loci allocated the Andaman samples (Satun, Trang, and Phangnga) into one cluster, and Chumphon and Trat into other clusters. Geographic differentiation between Satun-Trat and Satun-Phangnga was found only at the ZFP locus (P < 0.05), suggesting low degrees of genetic subdivision of Thai P. monodon.     

Low levels of genetic variation inPhenakospermum guyannense (Strelitziaceae), a widespread bat-pollinated Amazonian herb

Phenakospermum guyannense is a monotypic, arborescent, long-lived monocot that is widespread in Amazonian South America. This outcrossing species is pollinated primarily by phyllostomid bats. Given these life-history characteristics,P. guyannense is expected to exhibit high levels of genetic variation and gene flow. We used isozyme electrophoresis and randomly amplified polymorphic DNA (RAPD) to characterize genetic variation in populations ofP. guyannense from French Guiana. Both measures detected a surprisingly low level of genetic variation, with only five out of twenty (25%) allozyme loci polymorphic (P), 1.35 alleles per locus (A), and an expected heterozygosity (H_e) of 0.090 at the species level. Isozymic genetic variation was even lower within populations (P = 17.5, A = 1.24, H_e = 0.074), and was corroborated by a RAPD assay that used 26 arbitrary primers (P = 3.61, A = 1.04, H_e = 0.014). Although overall levels of variation were low, the detectable variation was distributed as would be expected for an outcrossing species with extensive gene flow (mean G_ST = 0.230). We suspect thatP. guyannense is depauperate in genetic variation because of a series of bottlenecks that affected the species over this portion of its range.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.