Karyotype and genome size estimation of Haliotis midae: estimators to assist future studies on the evolutionary history of haliotidae.
KEY WORDS: abalone, flow cytometry, karyotype, genome size, evolutionary history, speciation, Haliotis midae
Six abalone species inhabit the coastal waters of Southern Africa (Haliotis alfredensis, H. midae, H. parva, H. pustulata, H. queketti, and H. spadicea). These species, excluding H. pustulata, which also occurs along the coast of east Africa and southwest Asia, are endemic to South Africa. Only H. midae has been the subject of any cytogenetic work and has a chromosome number of 2n = 36 (Van der Merwe & Roodt-Wilding 2008).
Worldwide, the genus Haliotis is represented by 56 extant species (Fig. 1). Several of these abalone species are commercially cultured, and their economic value has promoted research including morphological, genetic, ecological, and physiological perspectives aimed at increasing the productivity of these cultured animals (Elliott 2000, Roodt-Wilding & Slabbert 2006). In addition to improvement programs, several studies also focused on investigating the origin and phylogeny of Haliotidae (e.g., Geiger & Poppe 2000, Estes et al. 2005, Streit et al. 2006). According to the current accepted view, the genus Haliotis originated in the ancient Tethys Sea and gave rise to 2 colonization events resulting in the modern distribution of the species (Fig. 1).
The karyotype has only been described in a few abalone species, and diploid numbers vary between 2n = 28, 2n = 32, and 2n = 36 (Table 1: Minkler 1977, Arai et al. 1982, Arai et al. 1988, Colombera & Tagliaferri 1983, Miyaki et al. 1997, Jarayabhand et al. 1998, Miyaki et al. 1999, Okumura et al. 1999, Hernandez-Ibarra et al. 2004, Gallardo-Escarate & Del Rio-Portilla 2007, Van der Merwe & Roodt-Wilding 2008, Carr & Appleyard 2009). In each of these, the fundamental number (NF) is double the diploid number because no telocentric chromosomes were observed. Although the use of various molecular markers have given new insights into the phylogenetic status of the genus, the mechanisms involved in chromosomal variability are not known. In fact, the chromosomal rearrangements that led to the three observed diploid numbers have not yet been investigated. Chromosomal rearrangements (e.g., deletions, inversions, centromeric fusion or fissions) are extensively studied and are known to generate karyotypic variation in populations of plants and animals (White 1978, Rieseberg & Willis 2007). Several studies have focused on chromosomal speciation in a variety of taxa (White 1978, Ayala & Coluzzi 2005, Rieseberg & Willis 2007, Hoffmann & Rieseberg 2008), but only a few have covered marine gastropods (Thiriot-Quievreux 1994, Pascoe et al. 1996, Amar 2003, Pascoe et al. 2004).
A useful tool to assess the genetic characteristics of a species and to understand the basis of evolutionary changes is genome size (C value) estimation. The evolutionary role of genome size is well studied within molluscs (Thiriot-Quievreux 1994, Thiriot-Quievreux 2003, Pascoe et al. 2004), but it is a complex topic because of the variability of genome sizes among eukaryotic organisms (it can vary by 5 orders of magnitude or more) (Cavalier-Smith 1982). The resolution of this issue, for Haliotis, will require the accumulation of data from additional abalone species covering the entire geographical distribution range.
A widely used method to determine the DNA content is flow cytometry. It is a quick and accurate way of estimating relative nuclear DNA content of large numbers of nuclei from different tissues (Vinogradov 1998, Dolezel et al. 2004, Dolezel & Bartos 2005), but has not been used for determining abalone genome size yet. The published haploid DNA content of the 3 Californian abalone species was calculated using fluorescence image analysis. Gallardo-Escarate et al. (2005) reported a haploid genome size of 1.77 pg in H. rufescens, and Gallardo-Escarate & Del Rio-Portilla (2007) reported the haploid genome sizes for H. corrugata of 2.14 pg, H. rufescens as 1.82 pg, and H. fulgens as 1.71 pg. The genome size data obtained by image analysis methods are comparable with flow cytometry data (Uozu et al. 1997).
In this study, flow cytometry was used to determine the genome size of the South African abalone H. midae by comparing hemolymph nuclei to the rainbow trout (Oncorhynchus mykiss) erythrocyte nuclei. Further cytogenetic analyses were also carried out to characterize the karyotype of H. midae in terms of chromosome arm length. The aim of this study is to contribute
to the knowledge of the Haliotis species, particularly the correlations of the previously mentioned parameters. The results will be discussed in the light of the results obtained for the 3 Californian abalone species (H. corrugata, H. rufescens, and H. fulgens), for which both genome size and karyotype composition are known.
[FIGURE 1 OMITTED]
MATERIALS AND METHODS
Genome Size Estimation
Genome size was estimated by flow cytometry using hemocyte nuclei of adult abalone (H. midae) and, as an internal standard, erythrocyte nuclei from the rainbow trout O. mykiss. The analysis was performed in triplicate and, from the results obtained, the mean value was calculated.
Collection and Storage of Genomic Material from Circulatory Cells
Abalone samples were obtained from the HIK Abalone Farm (Hermanus, South Africa). Hemolymph was drawn from the palial sinuses of abalone with a shell length of 150-200 mm using a 28-gauge needle and was collected into sterile 2-mL tubes. As a result of cell degradation after freezing, hemocytes were not frozen in the same manner as trout blood cells, but were immediately subjected to the staining protocol after collection. Hemolymph was sampled fresh for each replicate.
The trout blood was collected by cutting off the tail of a trout fingerling and bleeding the caudal vein into sterile 50-mL conical tubes. The blood sample was diluted with phosphate buffered saline and the blood cells were counted using a hemocytometer. Cells were then collected by centrifugation (2,000g, 10 min at 4[degrees]C). These cells were diluted to a concentration of 1.6 x [10.sup.8] cells/mL using the buffer solution from the CycleTEST PLUS DNA Reagent Kit (Becton Dickinson, Biosciences, San Jose, CA) and stored at -80[degrees]C in 1-mL aliquots. Trout blood cells stayed intact after freezing and did not have to be sampled fresh for each replicate.
Sample Preparation of Circulatory Cells for Flow Cytometry
The frozen trout blood samples were thawed on ice. Fresh abalone hemolymph and thawed trout blood were simultaneously centrifuged (1,000g, 5 min at room temperature) before CycleTEST PLUS (Becton Dickinson) solutions were added in succession according to the guidelines of the manufacturer. Propidium iodide, contained in one of these solutions, provided the basis of the stain. The samples were then filtered using 40[micro]m cell strainers (BD Falcon) and stored on ice until flow cytometry analyses, which needed to be performed within 2 h of sample preparation.
Flow Cytometry Analysis
All flow cytometry analyses were performed on a FACSAria flow cytometer (Becton Dickinson). It is equipped with a 585/42 bandpass filter used to detect propidium iodide-stained nuclei that emit at wavelengths ranging between 580 nm and 650 nm.
For each sample, population information from a minimum of 50,000 nuclear events was acquired using FACSDiva version 6.1 software. The same software was used to display data acquired from each nuclear population. The intensity of DNA intercalation in the cell was analyzed in a histogram data display using a logarithmic scale. The geometric mean fluorescence intensity of each population was used for further data analyses for comparison between populations.
Genome Content and Size Estimation
The 2C value for O. mykiss was taken as 5.5 pg (Hartley & Home 1985, Tiersch et al. 1989). The average of the peak means of the 3 analyses were used to calculate the genome content. Nuclear DNA content of H. midae was calculated as follows:
H. midae DNA content = (H. midae peak mean/ O. mykiss peak mean) x 2C DNA content of O. mykiss
Genome size was calculated using the formulae proposed by Dolezel et al. (2003):
H. midae genome size (bp) = (0.978 X [10.sup.9]) x H. midae DNA content (pg)
Following conventional hatchery procedures, adult specimens of abalone from the HIK Abalone Farm (Hermanus, South Africa) were induced to spawn and eggs fertilized. Hatching trochophore larvae at 12-14 h postfertilization were used for the preparation of the chromosome slides by adapting different protocols (Miyaki et al. 1997, Hernandez-Ibarra et al. 2004, Van der Merwe & Roodt-Wilding 2008). Briefly, larvae were concentrated and maintained in a 0.06% colchicine solution for 2 h in 1.5-mL tubes filled with autoclave-sterilized seawater. Hypotonic shock (KC1 solution 0.075 M for 70 min.) was applied to the larvae, followed by fixation in Carnoy solution (ratio of methanol to acetic acid, 3:1), exchanging this solution every 20 min, for a total of 4 times. Chromosome spreads were obtained by the dissociation of 30-50 larvae in 200 [micro]L 60% acid acetic. The suspension was dropped onto prewashed slides heated to 60[degrees]C and air-dried. Digital images of well-spread metaphase plates were captured using the Genus software (Applied Imaging Genetix, Queensway, UK). The best 10 plates with 2n = 36 were used for the karyotype analysis. These plates were selected from different slides to ensure that different individuals were represented. Relative arm length (short and long arms) of each chromosome was calculated from the total chromosome length (Thiriot-Quievreux 1984). Arm ratio (AR) and centromeric index (CI) was determined according to Levan et al. (1964). Classification of chromosomes was done using the nomenclature proposed by Levan et al. (1964) using the AR and CI values (M, metacentric; SM, submetacentric; ST, subtelocentric; T, telocentric). When the confidence intervals for either the AR or the CI (mean [+ or -] SD) overlapped the limit of the 2 different classes, chromosomes were classified as intermediates.
The haploid genome content of H. midae was calculated as C = 1.43 [+ or -] 0.02 pg, whereas the genome size was 2,803.68 [+ or -] 42.24 MB. The results indicate that H. midae has a smaller genome compared with H. rufescens, H. corrugata, and H. fulgens, even though the 4 species have the same chromosome number (2n = 36).
The karyotype of a representative metaphase plate is shown in Figure 2. After the decreasing measure of relative length, the chromosomes were numbered from 1-18. The relative chromosome length ranged from a maximum of 7.46 to a minimum of 4.22 (Table 2). Based on the AR and CI, the karyotype of H. midae is constituted of 6 pairs of metacentric, 10 pairs of submetacentric, and 2 pairs of subtelocentric (6M + 10SM +2ST) chromosomes. Only chromosome 5 has an AR/CI confidence interval that overlaps 2 classes, even though tending to the metacentric class considering the mean value and SD (AR = 1.71 [+ or -] 0.07, CI = 36.78 [+ or -] 1.08). For this reason, it was classified as metacentric-submetacentric (M-SM), which leads to the strict description of the karyotype as (6M + 1M-SM + 9SM + 2ST). No telocentric chromosomes were observed.
[FIGURE 2 OMITTED]
The estimation of genome size is important for its contribution in facilitating future genome research (Gregory et al. 2007). In the haliotids, for which the phylogenetic relationships and chromosomal evolution of the extant species are still to be clarified, it could be a useful tool for shedding new light on the evolutionary history of this genus. H. midae is only the fourth abalone species for which the genome size has been reported. The only other abalone species with reported genome sizes are the Californian species H. corrugata, H. rufescens, and H. fulgens (Gallardo-Escarate et al. 2005; Gallardo-Escarate & Del Rio-Portilla 2007). The South African abalone has a genome size of C = 1.43 pg; a low value when compared with those of the Californian species (C = 1.71-2.14 pg). The observed variability in DNA content is common for several taxa with a diploid structure, mainly because of the abundance of transposable elements and satellite DNA (Gregory et al. 2007). The form and function of the additional DNA is difficult to resolve without adequate comparative genome resources. All 4 species of abalone (including H. midae) have a diploid chromosome number of 36. Cytogenetic and corresponding DNA content for the Californian species suggest a positive correlation between genome size and the increase in metacentric/submetacentric chromosomes with a subsequent loss of submetacentric/subtelocentric chromosomes (H. corrugata: 10M + 7SM + 1ST, genome size 2.14 pg; H. rufescens: 8M + 9SM + 1ST, genome size 1.82 pg; H. fulgens: 8M + 8SM + 2ST, genome size 1.71 pg). The same pattern is seen for H. midae, where the smaller genome size (1.43 pg) is in agreement with the lower number of metacentric chromosomes and the higher number of subtelocentrics compared with the Californian abalone species. In the Californian abalones, the transition of 2 metacentric to 2 submetacentric chromosomes (H. corrugata [left and right arrow] H. rufescens) involves a difference in genome size of 0.32 pg. A similar difference in magnitude (0.38 pg) was observed between H. rufescens and H. midae, where the same number of M [left and right arrow] SM transitions occurred. The observed M SM [right arrow] ST transition with decreasing DNA content suggests that a loss of DNA, in the form of noncoding heterochromatic material, could be the most likely mechanism producing the genome size variability among the different abalone species. Heterochromatic polymorphisms were described in various taxa of plants and animals (Redi et al. 2001, Gregory et al. 2007) as the most common event generating karyotypic variability between closely related species and also providing some intraspecific variation (e.g., Garagna et al. 1999). Moreover, because of its high packing ratio (euchromatin and heterochromatin have respective packing ratios of 1,000 and 10,000 (Lewin 1999), the loss or addition of heterochromatic material could explain the large genome size difference among the studied abalone.
Overall, there appear to be no general rules concerning genome size and chromosomal rearrangements in the evolution of molluscs or other taxa (Thiriot-Quievreux 1994, Thiriot-Quievreux 2003, Pascoe et al. 2004; Pascoe 2006). The trend seen for the Haliotis spp. thus far can therefore only be interpreted in the light of the current evolutionary status and knowledge of this genus. Recent studies have investigated the evolutionary relationships of the haliotids using molecular markers (see, for example, Brown 1993, Lee & Vacquier 1995, Lee et al. 1995, Metz et al. 1998, Geiger 2000, Coleman & Vacquier 2002, Estes et al. 2005, Streit et al. 2006, Bester-Van der Merwe 2009). They agree in showing the existence of two monophyletic groups in Haliotidae: one grouping consisting of species from Europe, South Africa, Australia, Taiwan, and Japan, and the other containing Californian and Japanese species (Fig. 1). The results from the current study, in combination with the observation of geographical distribution of the Haliotis species with a known chromosome number (Fig. 1, Table 1), support the Tethys model of origin of Haliotidae (Geiger & Groves 1999) and also fits with the latest evolutionary model proposed by Estes et al. (2005). Following this model, the ancestral Haliotidae originated in the ancient Tethys Sea and gave rise to the modern Mediterranean abalone and to the tropical abalones of the Indian Ocean. Successively, two different lineages originated from these early tropical Haliotidae: the first one spread in a southeasterly direction and gave rise to the southern African, Australian, New Zealand, and tropical western Pacific species; whereas the second one, more recent, spread in a northeasterly direction and resulted in the North Pacific species (Californian and Japanese species).
Under this scenario, the fact that the most ancient species of the Mediterranean region have 2n = 28, the intermediate-age Indo-Pacific region species have 2n = 32, and the most recent species with 2n = 36 inhabit the Californian and the Japanese coast lead to the hypothesis that the evolutionary process of the abalone could occur with an increase in the chromosome number. Even if no general evolutionary trend in chromosome number evolution is evident in various invertebrate groups studied thus far, the hypothesis of an increase of chromosome number during the evolution process of abalone is supported in light of the basal gastropods and other Vetigastropodae having karyotypes with low diploid numbers equal to 2n = 18-20 (Haszprunar 1988). Conversely, the comparative values from 8 species of Muricidae, a widespread family of carnivorous marine gastropods, suggest a general evolutionary trend toward an increase in genome size and a decrease in chromosome number within the group (Pascoe et al. 2004). These contrasting findings warn against the use of chromosome number as an indicator of evolutionary relationships; a parameter that should be interpreted with caution in gastropods (Thiriot-Quievreux 1994, Thiriot-Quievreux 2003). Regardless, the recently described diploid chromosome number of 36 for H. midae (Van der Merwe & Roodt-Wilding 2008) and for the Australian H. rubra and H. laevigata (Carr & Appleyard 2009), both species belonging to the southeastern radiation, corroborate the chromosomal evolutionary trend predicted by the tropical and northern hemisphere abalone species (northward radiation species), for which the increase of chromosome numbers from 2n = 28 to 2n = 36 occurred. The fact that the same phenomenon is reported in 2 independent evolutionary lineages (the abalone belonging to the previously described colonization events, see Fig. 1) suggests that chromosomal rearrangements may result in species that characteristically have a high chromosome number. Because all the described haliotid karyotypes, however, show differences in both diploid and fundamental number, in the absence of comparative cytological data, it is impossible to speculate about the chromosomal rearrangements the genus might have undergone. We can only hypothesize that some complex rearrangements involving chromosomal fissions may have occurred.
Under the model discussed here, the somatic chromosome number of H. midae of 2n = 36 is not related to the 2n = 36 found in the North Pacific species. This could therefore lead to the hypothesis that some preferential chromosomal rearrangements are involved in the evolutionary process of abalone, but cytological data are required to verify this. Even though there is concordance of the pattern described by Gallardo-Escarate and Del Rio-Portilla (2007) with that observed in the South African abalone, this should be interpreted with caution because of the lack of comparative data (for instance, FISH experiments between chromosomes of the species belonging to the two different phylogenetic groups having the same diploid number) supporting this theory. On the other hand, even if this hypothesis has no strong foundation at this stage, its relevance could be stressed by the clarification of the mechanisms and functional importance of an increase in chromosomal numbers, the highest reported in the genus being 2n = 36. The reasons behind this event are yet unexplained, but it seems highly unlikely that the actual chromosome numbers of the species and the increased number observed is the result of a stochastic process. At this time, the selective forces driving such chromosomal rearrangements toward high diploid numbers and the role of the variation of genome size in the chromosomal evolutionary trends in Haliotidae can only be postulated.
Future studies on southern African abalone will include the determination of chromosome numbers, their karyotypic description, and genome size estimation to elucidate better the local evolutionary history of the genus. Furthermore, FISH experiments among species of the two proposed radiations will be necessary to clarify the mechanisms involved in the chromosomal rearrangements.
The authors thank Anvor Adams from the Jonkershoek hatchery (South Africa) for providing the trout samples, HIK Abalone (South Africa) for providing abalone samples, Ben Loos for his assistance with the flow cytometry analyses, Belinda Swart for technical assistance during the optimization of the karyotyping, Aletta Bester-Van der Merwe for commenting on some earlier drafts of this manuscript, and Stellenbosch University for the Use of facilities. This study was funded by the National Research Foundation.
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PAOLO FRANCHINI, RUHAN SLABBERT, MATHILDE VAN DER MERWE, ADELLE ROUX AND ROUVAY ROODT-WILDING *
Molecular Aquatic Research Group, Department of Genetics, Stellenbosch University, Private Bag XI, Matieland, 7602, South Africa
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Chromosome numbers and genome sizes (C values) published for the genus Haliotis based on the geographic distributions. Distribution Species 2n European- H. tuberculata 28 Mediterranean Asian Indo-Pacific H. varia 32 H. asinina 32 H. ovina 32 H. planata 32 H. exigua 32 H. diversicolor 32 African H. midae 36 Indo-Pacific H. rubra 36 H. laevigata 36 Pacific Northwest H. discus 36 hannai H. discus 36 discus H. madaka 36 H. gigantea 36 Pacific Northeast H. corrugata 36 H. cracherodii 36 H. fulgens 36 H. rufescens 36 Distribution Species Reference European- H. tuberculata Colombera & Tagliaferri Mediterranean (1983) Asian Indo-Pacific H. varia Jarayabhand et al. (1998) H. asinina Jarayabhand et al. (1998) H. ovina Jarayabhand et al. (1998) H. planata Arai et al. (1988) H. exigua Arai et al. (1988) H. diversicolor Arai et al. (1988) African H. midae Van der Merwe & Indo-Pacific Roodt-Wilding (2008) H. rubra Carr & Appleyard (2009) H. laevigata Carr & Appleyard (2009) Pacific Northwest H. discus Okumura et al. (1999) hannai H. discus Arai et al. (1982) discus H. madaka Miyaki et al. (1999) H. gigantea Miyaki et al. (1997) Pacific Northeast H. corrugata Gallardo-Escarate & Del Rio-Portilla (2007) H. cracherodii Minkler (1977) H. fulgens Hernandez-Ibarra et al. (2004) H. rufescens Hernandez-Ibarra et al. (2004) Distribution Species C Value (pg) European- H. tuberculata Undetermined Mediterranean Asian Indo-Pacific H. varia Undetermined H. asinina Undetermined H. ovina Undetermined H. planata Undetermined H. exigua Undetermined H. diversicolor Undetermined African H. midae 1.43 Indo-Pacific H. rubra Undetermined H. laevigata Undetermined Pacific Northwest H. discus Undetermined hannai H. discus Undetermined discus H. madaka Undetermined H. gigantea Undetermined Pacific Northeast H. corrugata 2.14 H. cracherodii Undetermined H. fulgens 1.71 H. rufescens 1.82 Distribution Species Reference European- H. tuberculata -- Mediterranean Asian Indo-Pacific H. varia -- H. asinina -- H. ovina -- H. planata -- H. exigua -- H. diversicolor -- African H. midae This study Indo-Pacific H. rubra -- H. laevigata -- Pacific Northwest H. discus -- hannai H. discus -- discus H. madaka -- H. gigantea -- Pacific Northeast H. corrugata Gallardo-Escarate & Del Rio-Portilla (2007) H. cracherodii -- H. fulgens Gallardo-Escarate & Del Rio-Portilla (2007) H. rufescens Gallardo-Escarate & Del Rio-Portilla (2007) TABLE 2. Relative length, arm ratio, centromeric index, and nomenclature following Levan et al. (1964) of the 18 chromosome pairs. Chromosome Relative Arm Pair Length [+ or -] SD Ratio [+ or -] SD 1 7.46 [+ or -] 0.29 1.26 [+ or -] 0.02 2 7.17 [+ or -] 0.09 1.41 [+ or -] 0.07 3 6.89 [+ or -] 0.02 1.91 [+ or -] 0.16 4 6.28 [+ or -] 0.02 1.81 [+ or -] 0.10 5 6.02 [+ or -] 0.06 1.71 [+ or -] 0.07 6 5.87 [+ or -] 0.19 2.20 [+ or -] 0.49 7 5.84 [+ or -] 0.04 1.89 [+ or -] 0.04 8 5.53 [+ or -] 0.02 1.24 [+ or -] 0.04 9 5.39 [+ or -] 0.02 2.68 [+ or -] 0.35 10 5.38 [+ or -] 0.08 1.27 [+ or -] 0.08 11 5.27 [+ or -] 0.04 2.09 [+ or -] 0.02 12 5.24 [+ or -] 0.12 1.88 [+ or -] 0.11 13 4.91 [+ or -] 0.01 1.04 [+ or -] 0.03 14 4.78 [+ or -] 0.08 1.10 [+ or -] 0.02 15 4.73 [+ or -] 0.02 1.75 [+ or -] 0.04 16 4.51 [+ or -] 0.14 2.20 [+ or -] 0.03 17 4.46 [+ or -] 0.10 4.56 [+ or -] 0.15 18 4.22 [+ or -] 0.03 4.20 [+ or -] 0.18 Chromosome Centromeric Pair Index [+ or -] SD Nomenclature 1 44.34 [+ or -] 0.10 M 2 41.44 [+ or -] 1.21 M 3 34.38 [+ or -] 1.79 SM 4 35.54 [+ or -] 1.28 SM 5 36.78 [+ or -] 1.08 M-SM 6 31.39 [+ or -] 3.87 SM 7 34.52 [+ or -] 0.46 SM 8 44.49 [+ or -] 0.80 M 9 27.02 [+ or -] 2.89 SM 10 43.98 [+ or -] 1.61 M 11 32.36 [+ or -] 0.12 SM 12 34.76 [+ or -] 1.39 SM 13 48.87 [+ or -] 0.91 M 14 47.70 [+ or -] 0.21 M 15 36.69 [+ or -] 0.48 SM 16 31.25 [+ or -] 0.02 SM 17 17.94 [+ or -] 0.47 ST 18 19.22 [+ or -] 0.66 ST M, metacentric; SM, submetacentric; ST, subtelocentric.
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|Author:||Franchini, Paolo; Slabbert, Ruhan; Van Der Merwe, Mathilde; Roux, Adelle; Roodt-Wilding, Rouvay|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2010|
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