Choanoflagellates are single-celled aquatic flagellates with a unique morphology consisting of a cell with a single flagellum surrounded by a ''collar'' of microvilli. They have long interested evolutionary biologists because of their striking resemblance to the collared cells (choanocytes) of sponges. Molecular phylogeny has confirmed a close relationship between choanoflagellates and Metazoa, and the first choanoflagellate genome sequence has recently been published. However, molecular phylogenetic studies within choanoflagellates are still extremely limited. Thus, little is known about choanoflagellate evolution or the exact nature of the relationship between choanoflagellates and Metazoa. We have sequenced four genes from a broad sampling of the morphological diversity of choanoflagellates including most species currently available in culture. Phylogenetic analyses of these sequences, alone and in combination, reject much of the traditional taxonomy of the group. The molecular data also strongly support choanoflagellate monophyly rejecting proposals that Metazoa were derived from a true choanoflagellate ancestor. Mapping of a complementary matrix of morphological and ecological traits onto the phylogeny allows a reinterpretation of choanoflagellate character evolution and predicts the nature of their last common ancestor.evolution ͉ morphology ͉ holozoa ͉ animals ͉ protists

Human activities are threatening Borneo's unique biodiversity, but little is known on the status of freshwater invertebrates. We assessed changes in diversity and distribution of freshwater mussels (Bivalvia: Unionida) in northern Borneo, and identified drivers of present distribution and threats. Past distribution data were collected from literature and museum resources. Present distribution data were collected from 21 river basins, and 47 water quality, climatic, landscape and human variables explored as potential predictors of species presence/ absence. Species delimitations were identified by morphology and COI barcoding, and haplotype networks generated. Our data indicate that over the past 50 years, four of originally five native species have become very rare or possibly locally extirpated. Since these four species are endemic to Borneo, other Bornean river basins should urgently be surveyed to identify any remaining populations. In the same time span, the non-native Sinanodonta woodiana has become the most widespread freshwater mussel in northern Borneo. The fifth native species was identified as Rectidens sumatrensis and found in four Sarawakian river basins, thus contradicting previous assumptions of an endemic Bornean Rectidens species. Although a number of stable R. sumatrensis populations are retained across Sarawak, the species' strong spatial contraction in mainland Sundaland and apparent low tolerance to eutrophication suggest that it is vulnerable to further habitat alteration. Our results indicate that Borneo's (endemic) freshwater invertebrate biodiversity is declining rapidly. Comprehensive surveys targeting an array of invertebrate and vertebrate taxa are needed to identify Borneo's freshwater biodiversity hotspots, where conservation efforts should be concentrated.

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Ex situ conservation of Malaysian Mahseer (Tor tambroides and T. douronensis), the ecologically threatened freshwater fish species in Malaysia via semen cryo-banking was started in 2008 with the mature Tor species from four different locations in Peninsular Malaysia. At current stage, a total of 150 ml semen sample was collected from male fish of 73 T. tambroides and 15 T. douronensis and kept cryo-storage in liquid nitrogen (LN 2 ) in the semen cryo-bank at Freshwater Fisheries Research Centre (FFRC). Sperm motility after cryo-preservation was found to be reduced by an average of 15 and 30% for T. douronensis and T. tambroides respectively; i.e. with prefrozen motility 87.8 ± 7.9% (n = 73) and 88.8 ± 6.7% (n = 15), and post-thaw motility 54.9 ± 14.3% (n = 73) and 74.0 ± 11.2% (n = 15) for T. tambroides and T. douronensis respectively. The mean fertilization and hatching rates by using cryo-preserved semen in T. tambroides were 36.4 ± 10.5% (n = 360) and 34.5 ± 11% (n = 360) respectively; whereas were 89.8 ± 10.2% (n = 360) and 80.2 ± 16.6% (n = 360) respectively for fresh semen. Meanwhile, the survival rate of fry produced from cryo-preserved sperm (97.6 ± 3.1%, n = 360) was equally good (P > 0.05) when compared to the use of fresh sperm (99.7 ± 0.5%, n = 360). Sperm motility (%) during milt collection, before freezing and after thawing of each cryo-preserved semen samples was recorded. These tests were done with sperm from all semen samples and kept up to date into the Fishery Information Network System (INFOFins). The subsequent fertilization capacity and hatching rates of eggs fertilized with sperm from selected cryo-preserved semen sample were also quantified and recorded into INFOFins. Future prospects of the semen cryo-banking of Malaysian Mahseer is also reported and discussed in the present paper.

Tomistoma schlegelii, the Freshwater Malayan Gharial or Tomistoma (local name: buaya jejulong), is secretive in nature; thus, very limited information is currently available on its wild population, especially in Sarawak. The objective of this study is to describe effort in assessing the Tomistoma wild populations in Ulu Sebuyau, Samarahan, and Serian, western Sarawak. Despite many challenges faced, this research has been successful at different levels. There was no Tomistoma seen during day surveys (2014-2015) in tributary of Samarahan River (Samarahan), Bunga-Baki River (Serian), and Kepayang River (Ulu Sebuyau). However, the research team came across two incidents of Tomistoma captured by the local people: (i) an adult male Tomistoma, captured in February 2015 in peat swamp area of Samarahan River tributary, and (ii) a hatchling, caught in Bunga-Baki River in March 2015. The sightings of Tomistoma in Kepayang River were reported by local people in February 2016; however, no individual was captured. The presence of hatchling in Bunga-Baki River indicates that a breeding population is most likely to occur in the area, and this gives hope for future conservation of Tomistoma in Sarawak. All sampling sites recorded almost similar water quality parameters and landscape of peat swamp areas, which previous studies claimed to be the potential Tomistoma habitats. The findings of this study should help relevant state agencies to step up efforts related to conservation of Tomistoma in Sarawak.

Eight single locus microsatellite markers were developed to characterize the Malaysian giant freshwater prawn, Macrobrachium rosenbergii. These microsatellites were isolated from an enriched genomic library contained by using a 5'-anchored polymerase chain reaction technique. Primers were designed to flank the repeat sequences and subsequently used to characterize 30 unrelated individuals of the giant freshwater prawn. The polymerase chain reaction amplification products of these eight microsatellite loci were polymorphic with the number of alleles ranging from two to 10 alleles per locus while the levels of heterozygosity ranged from 0.6333 to 0.8667.

Summary The objective of this study was to develop a cryopreservation method in Probarbus jullieni sperm, an endangered riverine fish species in Southeast Asia, including the optimization of an extender solution (14 extender formulations were tested) and selecting a cryoprotectant (five types of agents and methanol were used at concentrations (v/v) of 5, 7.5, 9, 10, 12, 15 and 20%). The semen to diluent ratios tested were as follow: 1 : 1, 1 : 2, 1 : 3, 1 : 4, 1 : 5, 1 : 7, 1 : 9, 1 : 14, 1 : 19, 1 : 24 and 1 : 49. Vapour exposure duration was set at 5, 10, 15 and 20 min while the distance between sample and liquid nitrogen (LN2) during the vapour exposure was designed at 3, 3.5, 4, 5 and 6 cm. Further, the time frame for thawing was set at 6, 7, 8, 10, 20 and 30 s. The optimum protocol was by using CF‐HBSS (pH 7.5, osmolality 285 ± 10 mOsmol kg−1) in combination with methanol at 9% (v/v); sperm to diluents ratio between 1 : 3 to 1 : 5; vapour exposure for 5 min or 10 min, with samples placed at 3.5 cm or 4 cm above LN2 and thawing at 40°C for 7 s. The mean of pre‐frozen and post‐thaw sperm motility was 80.1 ± 13.6% (n = 43) and 49.6 ± 16.4% (n = 43) respectively. The reproductive characteristics of P. jullieni during its spawning season were addressed in present work. Cryopreserved sperm was found to have lower fertilization ability (4.2 ± 2.5%, n = 1050) and hatching rate (1.6 ± 1.2%, n = 1050) compared with fresh sperm (fertilization 77.7 ± 6.2%, n = 1050; hatching 64.7 ± 7.7%, n = 1050). The resulted problems and constraints encountered in the process of sperm cryopreservation of the species studied were also reported in this paper.