Genetic differentiation of the Fejervarya limnocharis complex from Bangladesh and other Asian countries elucidated by allozyme analyses

The present study was conducted to elucidate the genetic divergence and the phylogenetic relationships in the F. limnocharis complex from Bangladesh and other Asian countries such as Sri Lanka, Thailand, Malaysia, Taiwan and Japan by allozyme analyses. We used a total of 95 frogs of the F. limnocharis complex from these countries and F. cancrivora from the Philippines as an outgroup. Based on body size, the F. limnocharis complex from Bangladesh was divided into three distinct groups: large, medium and small types. Allozyme analyses were carried out with 28 loci encoding 20 enzymes and two blood proteins by horizontal starch-gel electrophoresis. When genetic distance was calculated, distinct divergence was found among the three types: mean genetic distance was 0.782 between the small and medium types, 1.458 between the large and medium types, and 1.520 between the large and small types. Phylogenetic trees based on genetic distance showed that all populations of Bangladesh small type strongly formed a cluster and were found to be most closely related to the Sri Lanka population; that all populations of Bangladesh large type formed a very strong cluster and were grouped with several populations from Thailand, Malaysia, Japan, and Taiwan; and that the medium type was segregated from all other groups. This may imply that each of the three types is a different species, and that the medium type is possibly an undescribed taxon.     

Taxonomic status of three types of Fejervarya cancrivora from Indonesia and other Asian countries based on morphological observations and crossing experiments

Although the crab-eating frog Fejervarya cancrivora is one of the most widely distributed species in Asian region, taxonomic relationships among different populations remain unclarified. In this study, we attempted to elucidate the taxonomic status of F. cancrivora from Indonesian and other Asian populations. Five populations of F. cancrivora from Selangor (Malaysia), Cianjur (Java, Indonesia), Trat (Thailand), Khulna (Bangladesh), and Makassar (Sulawesi, Indonesia) were morphologically observed and subjected to crossing experiments. Principal component and clustering analyses revealed that these five populations could be organized into three groups corresponding to three observed morphological types: a Selangor and Cianjur group (large-type), a Trat and Khulna group (mangrove-type), and a Makassar group (Sulawesi-type). The limited crossing experiments revealed that hybrids between Selangor females and Cianjur and Trat males developed normally, whereas hybrids between Selangor females and Khulna males showed incomplete gametic isolation. Histological observations of the testes of mature males revealed the presence of pycnotic nuclei in the hybrids between Selangor females and Khulna males in addition to normal bundles of spermatozoa. In contrast, no pycnotic nuclei were observed in the Selangor controls. Although meiotic metaphases in the controls were normal, those in hybrids showed several abnormalities, such as the appearance of univalents and an increase in rod-shaped bivalents. Based on our findings from the morphological observations and crossing experiments, we conclude that each of three identified types represents a distinct species. We propose that the large-type is F. cancrivora, the mangrove-type is F. moodiei, and the Sulawesi-type represents an undescribed species.     

Morphological divergence, reproductive isolating mechanism, and molecular phylogenetic relationships among Indonesia, Malaysia, and Japan populations of the Fejervarya limnocharis complex (Anura, Ranidae)

In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0-0.4% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.     

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.     

Genetic divergences and phylogenetic relationships among the Fejervarya limnocharis complex in Thailand and neighboring countries revealed by mitochondrial and nuclear genes

To clarify the genetic divergence in the F. limnocharis complex from Thailand and neighboring countries and to elucidate the phylogenetic problems of this taxon, we analyzed partial sequences of the mitochondrial 12S and 16S rRNA genes and the nuclear CXCR4, NCX1, RAG-1, and tyrosinase genes. The F. limnocharis complex from Thailand had three distinct haplotypes for 12S and 16S rRNA genes. Nucleotide similarities and the phylogenetic relationships indicated that the haplotype 1 group corresponded to the real "F. limnocharis", the haplotype 2 group was F. orissaensis or closely related to it, and the haplotype 3 group was possibly an undescribed species. Mitochondrial gene data also showed two major clades of the genus Fejervarya, the Southeastern and South Asian groups. Although F. orissaensis is so far known only from Orissa in India, the haplotype 2 group was observed in Thailand. This distribution pattern and the phylogeny suggested that the origin of F. orissaensis and the haplotype 2 group might lie in Southeast Asia. There was also evidence suggesting that the haplotype 3 group originated in the South Asian area and has spread to northern Thailand. The nuclear gene data did not support the monophyly of the haplotypes recognized by mitochondrial genes. This incongruence between the mitochondrial and nuclear data seems to be caused by ancestral polymorphic sites contained in nuclear genes. Although neither the mitochondrial nor the nuclear data clarified intergeneric relationships, the nuclear data rejected the monophyly of the genus Fejervarya.     

Inter- and intraspecific evolutionary relationships of the rice frog Rana limnocharis and the allied species R. cancrivora inferred from crossing experiments and mitochondrial DNA sequences of the 12S and 16S rRNA genes

The rice frog Rana limnocharis is widely distributed in Southeast Asia and the rest of the Asian region extending from India to Japan. In Japan, the Sakishima-island populations of this species were regarded as a distinct species based on morphological and genetic divergences. The main purposes of this study were to confirm the presence of intraspecific reproductively isolating mechanisms in the Sakishima-island populations of R. limnocharis, and to clarify molecular inter- and intraspecific relationships of R. limnocharis and an allied species, Rana cancrivora. The hybridization experiments revealed that there were no reproductively isolating mechanisms between the Sakishima-island populations and other populations of R. limnocharis. The molecular evolutionary relationships were investigated by analyzing nucleotide sequences of the mitochondrial 12S and 16S rRNA genes using 12 populations of R. limnocharis from Japan and Taiwan, and two populations of R. cancrivora from Thailand and the Philippines. The phylogenetic trees constructed by the NJ method showed that the two populations of R. cancrivora were clearly separated from the 12 populations of R. limnocharis, and that the 12 populations of R. limnocharis were broadly divided into three clades; the first comprising eight populations from the main islands of Japan, the second comprising the Sakishima-island populations, and the third comprising the Okinawa-island and Taiwan populations. Interestingly, the Okinawa-island and Taiwan populations of R. limnocharis showed a close relationship that possibly reflected a secondary contact between the two populations. Based on the present crossing experiments and molecular data, it seems reasonable to regard the Sakishima-island populations as a single subspecies of R. limnocharis.     

A new species of the Fejervarya limnocharis complex from Japan (Anura, Dicroglossidae)

We describe a new species of dicroglossid frog of the Fejervarya limnocharis complex from western Honshu, Japan Mainland. The new species, Fejervarya kawamurai, is genetically closer to F. sakishimensis than to F. limnocharis. It differs from F. sakishimensis by smaller tympanum, head, forelimb, hindlimb, foot, and tibia lengths, all relative to snout-vent length, and from F. multistriata by relatively shorter forelimb, hindlimb, foot, and tibia. From F. limnocharis and F. iskandari, it is differentiated by relatively smaller forelimb, hindlimb, foot, and tibia lengths. Taxonomic problems of Fejervarya populations occurring in Central Ryukyus, continental China, and Taiwan are discussed.     

Allozyme analysis of cryptic species in the Asplenium niduscomplex from West Java,Indonesia

In various fern species, a large amount of rbcL sequence variation has been reported, and it is possible that these species contain several reproductively isolated cryptic species. In our previous study on Asplenium nidus L., it was suggested that the plants growing in Mt. Halimun National Park, West Java, Indonesia, consist of several cryptic species based on the results of crossing experiments among rbcL sequence types. In this study, we examined allozyme polymorphisms of five rbcL sequence types found in West Java in order to test the hypothesis that the assemblages of A. nidus delimited based on the rbcL sequences are separate Mendelian populations and gene flow is disrupted by reproductive isolation from one another. The calculated fixation indices suggested that the individuals in each rbcL type are randomly crossing at least in the investigated localities. Nevertheless, these rbcL-based assemblages were genetically differentiated in allozymes that are encoded in their nuclear genomes, and it is also suggested that gene flow is disrupted even between sympatrically distributed pairs of rbcL sequence types. Therefore, our findings support the view that the five rbcL sequence types in West Java are potential cryptic species. Electronic Publication     

Identifying hybridizing taxa within the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Müller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs.     

Phylogeography, genetic structure, and diversity in the dhole (Cuon alpinus)

The Asiatic wild dog or dhole was once very widely distributed across Asia but now has a very fragmented range. In this first genetic study of this little-known species, we obtained information on genetic diversity, phylogeography, and social structure using both mitochondrial control region sequencing and microsatellite genotyping of noninvasive faecal samples from wild populations, as well as from museum and captive samples. A pattern largely consistent with isolation by distance across the Asian mainland was observed, with no clear subspecies distinctions. However, two major phylogeographical groupings were found across the mainland, one extending from South, Central, and North India (south of the Ganges) into Myanmar, and the other extending from India north of the Ganges into northeastern India, Myanmar, Thailand and the Malaysian Peninsula. We propose a scenario involving glaciation events that could explain this pattern. The origin of the dhole populations in Sumatra and Java is enigmatic and requires further study. Very low levels of genetic diversity were observed among wild dholes from Baluran National Park in Java, Indonesia, but in contrast, high levels were observed in Mudumalai Wildlife Sanctuary in South India.     

Karyotypic differences of black rats,Rattus rattus,collected in various localities of East and Southeast Asia and Oceania

Karyotypes of several subspecies of black rats, Rattus rattus, collected in different localities of Asia and Oceania were examined with special emphasis on the relationship between the chromosome polymorphism and differentiation of the subspecies. Subspecies of black rats (R. rattus) collected were as follows; tanezumi from Japan; flavipectus and sladeni from Hong Kong; diardii, jalorensis from Kuala Lumpur, Malaysia; argentiventer from Kuala Lumpur, and Java and Celebes, Indonesia; mindanensis from Luzon and Mindanao, Philippines; and rattus from Australia, New Zealand, and New Guinea. Subspecies in Formosa, Korea and Thailand were not determined. All black rats collected in the above Asian districts had 42 diploid chromosomes, while those in Oceania had 38. The rats collected in Japan (tanezumi), Korea, Formosa, Thailand and Malaysia (diardii) had A/A No. 1 pair or polymorphic No. 1 (A/A, A/S and S/S) pairs, while those collected in Java and Celebes (argentiventer), Luzon and Mindanao (mindanensis) showed a higher frequency of S/S No. 1 pair. From the higher occurrence of No. 1 A/A pair of black rats in the Asian continent where the black rats originated, it is suggested that the original type of No. 1 chromosome pair of the black rats is A/A, and a pericentric inversion occurred in the acrocentric No. 1 chromosome and thus rats with subtelocentric No. 1 pair formed.—Black rats with 38 chromosomes were observed in Australia, New Guinea and New Zealand. These karyotypes seem to have developed by Robertsonian fusion of 4 acrocentric pairs (No. 4 and 7, and No. 11 and 12) in black rats of the Asian type. A relationship between body size and chromosome constitution was observed in subspecies of the black rats.Contribution No. 831 from the National Institute of Genetics, Japan. Supported by a grant-in-aid from the Ministry of Education of Japan (Scientific Expedition in 1968, No. 8801 in 1969, and No. 9001 in 1970).     

A redescription ofDiaphanosoma modigliani Richard, 1894 (Crustacea : Daphniiformes : Sididae)and an evaluation of its occurence outside Lake Toba (Sumatra)

A pelagic species of Diaphanosoma, D. modigliani is redescribed on material from the type locality (Lake Toba, Sumatra, Indonesia). Other records of this species in tropical Asia (India, Philippine Islands, Malaysia, Sri Lanka, Thailand) are either unsubstantiated or erroneous.     

Natural hybridization between Vanilla claviculata (W.Wright) Sw. and V. barbellata Rchb.f. (Orchidaceae): genetic, morphological, and pollination experimental data

Vanilla claviculata (W.Wright) Sw. and K barbellata Rchb.f. grow sympatrically in the southwestern part of herto Rico. At three localities in the range of the two species, individuals with flowers that appeared Merent from previously known species were found. To test whether these were hybrids between V. claviculata and V. barbellata , 11 morphological floral characters were recorded at seven populations and allozyme profiles of the three taxa compared. Principal component analysis on the morphological characters gave three distinct groups with minor overlaps. The first component axis reflects variation in overall size of the flowers, while the second component reflects variation in the size of the distal aperture of the labellum. Allozyme data revealed that the putative hybrids have a significant surplus of heterozygotes ( F _IS = - 0.65) as expected in a F ₁ generation. Both data sets support the hypothesis that the individuals are hybrids, which is further augmented by interspecific pollination experiments between the putative parental species. This is the first case of natural hybridization reported in the genus Vanilla .     

西北太平洋海岸带大弹涂鱼复合体的隐存种与进化历史

大弹涂鱼Boleophthalmus pectinirostris间断分布于西太平洋海岸带东亚与马来西亚马六甲海峡,但马来西亚种群的分类地位尚存争议。研究使用线粒体ND5基因序列（718 bp）与核位点Rag1基因序列（1395 bp）对西北太平洋海岸带11个地点的45尾大弹涂鱼属鱼类进行系统发育关系重建,结果表明大弹涂鱼包括东亚与马来西亚两个单系群,两者形成姊妹群关系。GMYC分析、*BEAST物种树支持大弹涂鱼东亚谱系和马来西亚谱系是不同种。分子测定年龄分析表明大弹涂鱼东亚谱系与马来西亚谱系之间的分化时间为2.73百万年。因此,西北太平洋海岸带大弹涂鱼是复合体,包括两个物种:东亚种群是大弹涂鱼Boleophthalmus pectinirostris sensu stricto,而马来西亚种群是隐存种Boleophthalmus sp.。大弹涂鱼与隐存种之间的物种分化可能是晚上新世冰期海平面下降产生的地理隔离以及间冰期洋流对基因交流的阻碍两方面相互作用的结果。       

Genetic Divergence among Southeast and East Asian Populations of Rana limnocharis (Amphibia: Anura), with Special Reference to Sympatric Cryptic Species in Java

An electrophoretic survey was conducted for the Southeast and East Asian populations of Rana limnocharis. Results indicated that specimens from a single locality in Java derived from two genodemes showing complete allelic displacement at eight out of 25 presumptive loci examined. Within each of these genodemes, genotype frequencies at all polymorphic loci were well concordant with the Hardy-Weinberg expectation. These results strongly suggest that those specimens actually represent two biological species. Comprehensive distance analyses suggested that one of the two species from Java is genetically most divergent among all samples examined, whereas the other is most similar to the Laos sample and then to a group consisting of samples from Hongkong, western China, and the southern Ryukyus. Large values of genetic distances obtained for each combination of allopatric samples imply the presence of additional cryptic species in this nominal species.     

SPINNER DOLPHINS (STENELLA LONGIROSTRIS) OF THE WESTERN PACIFIC AND SOUTHEAST ASIA: PELAGIC AND SHALLOW-WATER FORMS1

A dwarf form of the spinner dolphin has been reported from the Gulf of Thailand, while more typical large spinner dolphins have been described from Japanese waters and other localities in the western Pacific. These reports have been based on very few specimens. Our purpose in this study was to determine the affinities of spinner dolphins throughout the region based on larger samples and to review their taxonomic status, with an hypothesis of two widespread ecotypic forms, or subspecies. We examined 213 osteological specimens, from a tuna gillnet fishery in the Philippines, from the former Taiwanese shark gillnet fishery in the Timor and Arafura Seas off northern Australia, from the Gulf of Thailand, from other areas in the western Pacific and Southeast Asia, from the eastern Indian Ocean, and from the Central and South Pacific. Results show that spinner dolphins from the deep inner waters of the Philippines conform to the large pelagic type of spinner dolphin that inhabits the Central and South Pacific, the western Pacific and the eastern Indian Ocean. The skull is similar in size and shape to the holotype specimen of S. longirostris (from unknown locality). This form feeds primarily on small mesopelagic fishes and squids. Spinner dolphins from the shallow waters of inner Southeast Asia represented in the sample, including the Gulf of Thailand, Timor Sea and Arafura Sea, are smaller in body and skull size, have fewer teeth and vertebrae, and feed mainly on benthic and coral reef fishes and invertebrates. We hypothesize that this form also inhabits the Java Sea and other shallow waters throughout inner Indonesia and Malaysia. We redescribe a subspecies corresponding to the small form and based on Delphinus roseiventris Wagner 1846 from the Arafura Sea, designating a neotype and paraneotype specimens.     

Biogeographic variation in coral species diversity on coral reefs in three regions of Indonesia

Abstract . This paper assesses variation in coral species diversity within the Indonesian archipelago, and the influence of regional species pools, geomorphology and anthropogenic pollution on coral species diversity and occurrence. We obtained transects from 33 sites on 14 reefs in three regions of Indonesia: Ambon (Moluccas), South Sulawesi and the Java Sea. We determined the within‐site species richness by using species‐sampling curves. Cluster analysis and multi‐dimensional scaling showed that land‐based pollution was the primary determinant of coral species diversity and species occurrence on reefs. Relatively unaffected reference sites in eastern Indonesia were approximately 20% more diverse than Java Sea reference sites. Rare species formed a higher proportion of the coral fauna on eastern Indonesian sites, and eastern Indonesian apparent endemic species contributed approximately 25% of the total species pool sampled. Between‐site variation in species occurrence was lower on Java Sea reefs than on eastern Indonesian reefs. A larger species pool in eastern Indonesia than in the Java Sea probably accounted for most of the difference in within‐site species diversity between eastern Indonesian and Java Sea reference sites. High fishing intensity in the Java Sea, including destructive fishing practices, may have also contributed to reduced within‐site species diversity on Java Sea reference reefs. Despite the fact that the Java Sea was exposed during Pleistocene lowstands, and was recolonized by marine organisms only within the last 10 000 years, coral species diversity and assemblage composition on the Java Sea reefs was largely similar to open ocean reefs in eastern Indonesia.     

Australian Laurencia majuscula (Rhodophyta,Rhodomelaceae) and the Brazilian Laurencia dendroidea are conspecific

Morphological and molecular studies have been undertaken on two species of the red algal genus Laurencia J.V.Lamouroux: Laurencia majuscula (Harvey) A.H.S. Lucas and Laurencia dendroidea J.Agardh, both from their type localities. The phylogenetic position of these species was inferred by analysis of the chloroplast‐encoded rbcL gene sequences from 24 taxa. In all phylogenetic analyses, the Australian Laurencia majuscula and the Brazilian L. dendroidea formed a well‐supported monophyletic clade within the Laurencia sensu stricto. This clade was divided into two subclades corresponding to each geographical region; however, the genetic divergence between Australian L. majuscula and Brazilian L. dendroidea was only 0–1.35%. Examination of the type specimens and sequences of freshly collected samples of both Laurencia majuscula and L. dendroidea show the two to be conspecific despite their disjunct type localities.     

The ecology and biology of Southeast Asian false spider crabs (Crustacea: Decapoda: Brachyura: Hymenosomatidae)

The false spider crabs of the family Hymenosomatidae are one of the most poorly known group of brachyuran crabs in Southeast Asia. This is largely attributed to their small size and cryptic behaviour. Despite the many studies of decapods in Southeast Asia, only eleven species of hymenosomatids are known. Of these, two genera and six species were only described in the last two years. From Singapore the largest number of species (five) has been reported probably because it has been the best explored, while from Thailand three species and Peninsular Malaysia two species are known. There are no published records of hymenosomatids from Borneo or Java. The hymenosomatid fauna is perhaps best known in Australia and New Zealand. It is anticipated that when proper collections are made and studies implemented, the Southeast Asian hymenosomatid fauna will prove to be more diverse than what has been reported. The ecology of the Southeast Asian species is reviewed, especially in the view that two species are completely freshwater, one of which is a troglobite. Most species are littoral or sublittoral in habit and very sensitive to human activities (e.g. pollution).     

Reproductive isolation and hybrid pollen disadvantage in Ipomopsis

One cause of reproductive isolation is gamete competition, in which conspecific pollen has an advantage over heterospecific pollen in siring seeds, thereby decreasing the formation of F1 hybrids. Analogous pollen interactions between hybrid pollen and conspecific pollen can contribute to post-zygotic isolation. The herbaceous plants Ipomopsis aggregata and I. tenuituba frequently hybridize in nature. Hand-pollination of I. aggregata with pollen from F1 or F2 hybrids produced as many seeds as hand-pollination with conspecific pollen, suggesting equal pollen viability. However, when mixed pollen loads with 50% conspecific pollen and 50% hybrid pollen were applied to I. aggregata stigmas, fewer than half of the seeds had hybrid sires. Such pollen mixtures are frequently received if plants of the two species and F1 and F2 hybrids are intermixed, suggesting that this advantage of conspecific over hybrid pollen reduces backcrossing and contributes to reproductive isolation.