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Genome size (C-value) is a fundamental characteristic of every species and is very important for the progress of molecular cytogenetic and phylogenic studies and for whole-genome sequencing. Changes in DNA quantity by genome duplication leading to the emergence of new functional genes have played an important role in genome evolution (Holland et al. 1994, Ohno 1999). In the animal kingdom, variation of C-values differs widely, ranging from 18.8 Mbp (~0.02 pg) in the banana root nematode Pratylenchus coffeae (Leroy et al. 2007) to 132.83 pg in the African lungfish Protopterus aethiopicus (Pedersen 1971). The C-value is usually similar among closely related species, but there are some exceptions. In plants, for example, differences in C-values have been reported within Hydrangea sp. inhabiting different regions and between subspecies [e.g., Hydrangea macrophylla (Thunb.) ser. subsp. macrophylla McClint. and H. macrophylla (Thunb.) Ser. subsp. serrata (Thunb.) Makino] (Cerbah et al. 2001). Another important genomic feature is the percentage of adenine and thymine nucleotides (AT content). In the genus Festuca, for example, it was reported that the proliferation of GC-rich retrotransposons in the genome of basal species with the subsequent reduction of the C-value and GC content was related to the evolution of this genus (Smarda et al. 2008). The existence of C-value differences in closely related species inhabiting different regions, and relationship between C-value and cell characteristics, such as GC%, nucleus, and cell sizes and rates of mitosis has been reported by some authors, which is called the "C-value enigma" (Vinogradov 1998, Gregory 2001). Yet, the evolutionary role of nucleotide rate differences in lower taxonomic groups within the animal kingdom is still unknown.

The family Haliotidae contains approximately 70 species and is widely distributed in oceans and seas surrounding Europe, Africa, Asia, Oceania, and in the North Pacific (Abbott & Dance 1985). Haliotidae most likely originated from abalones inhabiting the ancient Tethys Sea and based on chromosome number (the Tethys model), the family is classified into two different lineages (Geiger & Groves 1999, Franchini et al. 2010, Bester-van der Merwe et al. 2012). The Indo-Pacific lineage comprises South African, Oceanian, and tropical Western Pacific species, all of which possess 2n = 32 or 36 chromosomes (Nakamura 1985, Arai et al. 1988, Jarayabhand et al. 1998, Geiger & Groves 1999, Franchini et al. 2010, Arai & Okumura 2013, Botwright 2015). The North Pacific lineage comprises North Pacific species, all of which have In = 36 chromosomes (Minkler 1977, Arai et al. 1982, Miyaki et al. 1997, Geiger & Groves 1999, Miyaki et al. 1999, Gallardo-Escarate & Del Rio-Portilla 2007, Arai & Okumura 2013). Yet, studies on the C-value (or amount of DNA contained within haploid nucleus) of this genus within each geographic area are fewer than chromosomal studies. For the North Pacific lineage, C-values have been reported for three species, Haliotis rufescens (Swainson, 1982) (1.82 pg), Haliotis corrugata (Wood, 1828) (2.14 pg), and Haliotis fulgens (Philippi, 1845) (1.71 pg) (Gallardo-Escarate & Del Rio-Portilla 2007). For the Indo-Pacific lineage, C-value was only reported for Haliotis midae L. (1.43 pg), a South African species (Franchini et al. 2010). In a previous study, the C-values of Haliotis discus hannai (Ino, 1953) (1.84 pg) from Northwest Pacific and Haliotis diversicolor aquatilis (Reeve, 1845) (1.45 pg) from Southeast Asia were determined (Adachi & Okumura 2012). Based on the 1.25-fold variation found in the C-values of three Haliotis species from Northeast Pacific (Gallardo-Escarate & Del Rio-Portilla 2007), variation in C-values are expected within and between other geographic areas. Despite the differences in chromosome number between the North Pacific and Indo-Pacific lineages, increases in C-values related to increases in chromosome number might not be detected between these areas because C-values and chromosome numbers are not necessarily proportional (Rodriguez-Juiz et al. 1996, Gregory & Hebert 2002, Pascoe et al. 2004). Because there is only one species within each area, C-value variations within a geographic area have not been studied so far and the relationship between chromosome numbers and C-values within each lineage are still not clear.

Building on previous work to examine chromosomes of abalone (Arai et al. 1982, Arai &Wilkins 1986, Arai et al. 1988, Okumura et al. 1999), the present study aims to further examine the chromosomal evolution within the genus Haliolis by increasing the knowledge on the C-value and AT content of five Southeast Asian and Northeast Pacific species and three Oceanian species, which represent a new geographic area that is less explored. Variations in C-values and AT content were evaluated in the context of geographic radiation in the chromosomal evolution of Haliotis.


Abalone Specimens

Sixteen Haliotis gigantea (Gmelin, 1791) and nine Haliotis madaka (Habe, 1977) specimens were collected by fishermen in April 2014 and March 2015 in the area around Awaji Island, Seto Inland Sea, Hyogo Prefecture, Japan. Thirteen Haliotis asinina L., four Haliotis varia L., and three Haliotis planata (G. B. Sowerby II, 1882) specimens were obtained from Aquaculture Department of Southeast Asian Fisheries Development Center in Tigbauan, Iloilo, Philippines in June 2012. Nine South African Haliotis midae specimens were obtained through a distributor in Japan in May 2012 (TOSENBO Co., Ltd, Chiba, Japan). Twenty Black lip Haliotis rubra (W. E. Leach, 1814) specimens were collected around Tasmania Island, Australia, in September-November 2014. Ten Green lip Haliotis laevigata (Donovan, 1808) specimens were collected around Kangaroo Island, Australia, at 9-35 m depth in February 2016. Twenty-one Haliotis roei (Gray, 1826) specimens were collected around Perth, West Australia, in November 2014. All Australian abalones were collected by local fishermen through a wholesaler in Japan (Tosenbo Co., Ltd). Shells were removed using a spatula and the mantle tissue of each individual was dissected and stored in Carnoy's fluid (methyl alcohokacetic acid, 3:1) at -20[degrees]C for 24 h. The geographical classification of abalones followed Geiger and Groves (1999), and Franchini et al. (2010).

C- Value and A T Content Analysis

The C-value and AT content of abalones were estimated using two different fluorescent dyes; propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) in PA-1 model flow cytometer (Partec Co., Muenster, Germany). Samples of about 3 mm were cut from each mantle tissue and crushed in two to three drops of solution A (CyStain DNA 2-step nuclei extraction buffer, Partec Co.). Estimation of C-value was based on PI measurements. A 700 [micro]L staining buffer (Partec Co.) was added to the cell suspension and the mixture was filtered through a 50 [micro]m CellTrics filter (Partec Co.) before adding 1.5 [micro]L RNase A and 3 [micro]L PI Stock Solution (Partec Co.). The solution was then incubated at room temperature for 30 min. For DAPI measurements, 500 [micro]L of solution B (CyStain DNA 2-step staining solution, Partec Co.) containing DAPI was added to the cell suspension and the mixture was filtered through a 50 [micro]m mesh. Chicken red blood cells (CRBC) (Nippon Bio-Test Laboratories Inc., Tokyo, Japan) and mantle cells of Haliotis discus hannai obtained from the hatchery of Kitanihon Fishery Co. (Iwate, Japan) that have C-values of 1.25 and 1.84 pg, respectively, were used as standards to minimize histogram measurement error as the distance between standard and target cells are difficult to distinguish. Measurements were based on at least 5,000 nuclei.

The C-value of each sample was calculated as the difference between the fluorescence intensity (FL) of standard and target cells (Fig. 1) according to Eq. 1 of Rees et al. (2008). Genome size (Mbp) was then obtained from the calculated C-value following Dolezel et al. (2003) Eq. 2. The AT content was calculated as the ratio between DAPI- and Pi-based FL following Godelle et al. (1993) Eq. 3.

C-value(pg) = (T F[L.sub.PI]/S F[L.sub.PI]) x [C-value.sub.standard], (1)

Genome size (Mbp) = (0.978 X [10.sup.9]) X C-value, (2)

AT(%) = [AT%.sub.standard]([[R.sub.DAPI]/ [R.sub.PI]).sup.1/3] (3)

where, T FL and S FL are the FL of target and standard cells, respectively. [C-value.sub.standard] is the C-value of CRBC (1.25 pg) or Haliotis discus hannai (1.84 pg), and [R.sub.DAPI] = T F[L.sub.DAPI]/S F[L.sub.DAPI] and [R.sub.PI] = T F[L.sub.PI]/S F[L.sub.PI]. Indices DAPI and PI correspond to the two fluorescent dyes used.

Statistical Analyses

Average C-values between geographical groups of abalones were analyzed by one-way analysis of variance (ANOVA) followed by Tukey-Kramer tests to determine the significance among them. Before analyses, the homogeneity in the variance of data was examined using the Bartlett test. Statistical analyses were conducted in BellCurve for Excel software (Social Survey Research Information Co., Ltd, Tokyo, Japan), considering P < 0.05 as the significance threshold.


Chromosome number, C-value, and AT content of each Haliotis species examined in the present study are shown in Table 1. In the present study, C-values ranged from 1.32 pg for Haliotis varia to 2.01 pg for Haliotis laevigata. Significant differences in C-values were detected among regions (Northwest Pacific, Northeast Pacific, Indo-Pacific, and Oceania; Bartlett test, P = 0.41; ANOVA test, P = 0.001). Within the Pacific Northwest groups, C-values ranged from 1.73 pg for Haliotis gigantea to 1.89 pg for Haliotis madaka. There were no significant differences (Tukey-Kramer test, P = 0.91) between the C-values of Northwest Pacific abalones (H. madaka, H. gigantean, and Haliotis discus hannai) and that of Northeast Pacific abalones (Haliotis corrugate, Haliotis rufescens, and Haliotis fulgens) (Table 1, Fig. 2). On the contrary, C-values within the Indo-Pacific lineage ranged from 1.32 pg in H. varia to 2.01 pg in H. laevigata. The South African species Haliotis midae presented a slightly higher value (1.49 pg) than that previously reported by Franchini et al. (2010) (1.43 pg); this value was within the range obtained for Asian Indo-Pacific species (from 1.32 pg for H. varia to 1.52 pg for Haliotis planata). Oceanian abalones (Haliotis rubra, H. laevigata, and Haliotis roei) presented significantly higher C-values than other Indo-Pacific abalones (Haliotis asinina, H. varia, H. planata, Haliotis diversicolor aquatilis, and H. midae) (Tukey-Kramer test, P = 0.003, Table 2, Fig. 2). Overall, the Indo-Pacific group, except Oceanian abalones, presented significantly lower C-values than Pacific Northwest (Tukey-Kramer test, P = 0.01) and Northeast (Tukey-Kramer test, P = 0.003) abalones (Table 2, Fig. 2).

The AT content ranged from 58.0% in Haliotis planata to 66.3% in Haliotis diversicolor aquatilis, corresponding to an 8.3% variation. The highest and lowest AT content was obtained for Indo-Pacific species (Table 1). This high variation in AT contents within the Indo-Pacific lineage is also evident in comparison with other areas; within the North Pacific (Pacific Northwest) and Oceanian groups, AT contents vary by 3.7% (from 62.3% in Haliotis discus hannai to 66.0% in Haliotis gigantea) and 1.4% (from 64.7% in Haliotis roei to 66.1% in Haliotis laevigata), respectively (Table 1). Nonetheless, no significant difference was detected among the AT contents of the several geographic groups (Bartlett test, P = 0.22; ANOVA test, P = 0.30).


This study evaluated the variations of C-value and AT content among Haliotis spp. from the Indo-Pacific to understand how chromosomal and genome size evolution has proceeded within the context of the recent geographic radiation of this species. The earliest abalones originated in the ancient Tethys Sea and then spread eastward, forming the Indo-Pacific lineage (Geiger & Groves 1999, Estes et al. 2005). Within this group, significantly different C-values were shown between Southeast Asian (Asian Indo-Pacific) and Oceanian (Oceanian Indo-Pacific) abalones, which might be related by their different chromosome numbers: 2n = 32 and 36, respectively (Table 1) (Nakamura 1985, Arai et al. 1988, Jarayabhand et al. 1998, Arai & Okumura 2013, Botwright 2015). The lower C-value in Asian Indo-Pacific species (2n-32) might reflect the lower C-value of earliest abalones. For example, the chromosome number of the African abalone Haliotis midae (2n = 36; Franchini et al. 2010) is higher than that of Asian Indo-Pacific abalones, but it is nested within the range found for Southeast Asian species (from 1.32 pg in Haliotis varia to 1.52 pg in Haliotis planata). The karyotype of H. midae (12m + 2m/sm + 18sm + 4st) [metacentric (m), submetacentric (sm), and subtelocentric (st) chromosomes] reported by Franchini et al. (2010) comprised more subtelocentric chromosomes than that found for the four Southeast Asian species (18m + 12sm + 2sm/st, and 16m + 16sm for H. varia, 18m + 12sm + 2sm/st for H. planata, 16m + 10sm + 4sm/st + 2st for Haliotis diversicolor aquatilis, and 20m + 12sm for Haliotis asinina) (Nakamura 1985, Arai et al. 1988, Jarayabhand et al. 1998, Arai & Okumura 2013). Generally, the fundamental number (FN), which is the number of visible major chromosomal arms per set of chromosomes, is an important component of the karyotype and is significant in understanding karyotype evolution--FN typically increases with chromosome fission, and decreases when fusion occurs (Mariano et al. 2012). Given that FN is two for biarm chromosome [metacentric (m) and submetacentric (sm) chromosomes] and one for monoarm chromosome [subtelocentric (st) and telocentric (t) chromosomes], FN was higher in H. midae than in Southeast Asian species (Table 1, Fig. 3). Furthermore, average chromosome size (C/n value, i.e., DNA content per haploid chromosome number) was relatively lower in H. midae compared with Southeast Asian species (Table 1, Fig. 3). Overall, these findings support the idea that chromosome number of H. midae increased by chromosome fission because the increase in chromosome number was not accompanied by increase in DNA amount. Moreover, it was observed that the Southeast Asian species had a higher C/n value in comparison with H. midae. In addition, the C-values of Haliotis rubra, Haliotis laevigata, and Haliotis roei, which spread toward the southern hemisphere along with H. midae, were higher than that of the other five Indo-Pacific species. The number of chromosomes in H. rubra and H. laevigata is 2n = 36 (Botwright 2015), as in H. midae. This increase in chromosome number seems not to be due to chromosome fission, as reported for H. midae, because the C-values and C/n value of Oceanian Indo-Pacific species is higher than those of H. midae and Southeast Asian species (Figs. 1 and 3). A recent phylogenetic study suggested a relatively recent radiation of the South African species, including H. midae, and that South African and Australian groups are sister clades (Bester-van der Merwe et al. 2012). Thus, the increase of chromosome number and concomitant DNA amount shown in Oceanian abalones would be relatively recent. Future study should examine the chromosome number, karyotype, and C-values of other South African species [e.g., Haliotis spadicea (Donovan, 1808), Haliotis alfredensis (Bartsch, 1915), Haliotis parva L., and Haliotis queketti (E. A. Smith, 1910)], to evaluate if the similarity of C-values observed between Southeast Asian species and H. midae is maintained for other South African species and if chromosome fission occurred only in H. midae.

Two major lineages within Haliotis, North Pacific and Indo-Pacific, were suggested by some authors (Coleman & Vacquier 2002, Estes et al. 2005, Streit et al. 2006, Bester-van der Merwe et al. 2012). This study supported previous studies reporting high C-values in the North Pacific group (Gallardo-Escarate & Del Rio-Portilla 2007, Adachi & Okumura 2012), and a significantly higher C-value was found in North Pacific species compared with Southeast Asian and South African species. Evidence of chromosomal evolution in the North Pacific lineage, such as increasing of C-value, number of chromosomes, and FN (Table 1), was also found in the aforementioned Haliotis rubra and Haliotis laevigata, which have spread toward the southern hemisphere. On the contrary, no significant differences in C-values were found between the two regions in the North Pacific group (Pacific Northeast and Northwest), although slight differences in species karyotypes were observed between the two areas. This suggested, possibly, that the significant increase in C-values was related to the increase in chromosome number observed among Southeast Asia, South African, and Oceanian species, which did not occur within the North Pacific group. Yet, slight differences in the karyotype of North Pacific species were detected. The karyotype of Haliotis discus hannai (20m + 16sm or 22m + 14sm; Arai et al. 1982, Okumura et al. 1995, 1999), Haliotis gigantea (20m + 16sm; Miyaki et al. 1997), and Haliotis madaka (20m + 16sm; Miyaki et al. 1999), which inhabit the Northeast Pacific comprised only m and sm chromosomes, whereas Haliotis rufescens (16m + 18sm + 2st), Haliotis corrugata (20m + 14sm + 2st), and Haliotis fulgens (16m + 16sm + 4st) inhabiting the Northwest Pacific, have st chromosomes (Gallardo-Escarate & Del Rio-Portilla 2007) and show higher variation in karyotypes than Northeast Pacific species. Within regions, the C-value of the three Northwest Pacific species increased less (1.09-fold) than that of Northeast Pacific species (1.25-fold). Phylogenic studies revealed that the North Pacific lineage diverged fairly recently from one ancestral type and spread across the Northeast Pacific (Coleman & Vacquier 2002, Estes et al. 2005): Northeast Pacific species then might have spread to the Northwest Pacific. The slightly different C-values found among closely related species might have been caused by small variations in the genome accumulated during chromosomal evolution. It would be usually small in a new area because variations of C-value and/or chromosomal mutation accumulate over time. Therefore, the low variation in karyotype and low expansion of C-values in Northeast Pacific species might be related to their radiation within a new geographical area.

The high C-values shown by the North Pacific lineage and Oceanian species suggests that some major evolutionary event resulted in the increase of DNA amount, an event that likely occurred independently in the ancestor of each of the different lineages. One possibility is that transposable elements are responsible for a large increase in the genome size over a relatively short period, similar to polyploidy, but this expansion did not apparently result in an increase of chromosome number. Similar evolutionary events that increased DNA amount by expansion of transposable elements, have been reported in other taxa. In grasses, for example, a large portion of the genome is made up of transposable elements (Meyers et al. 2001, Li et al. 2004). In a marine species, octopus Octopus bimaculoides, it was reported that 45% of the genome is composed of high-copy repetitive sequence, which was caused by two extensions of the transposon (e.g., retrotransposons) (Albertin et al. 2015, Mita & Boeke 2016). Therefore, expansion of transposon elements in the genome of North Pacific and Oceanian species may have occurred in these species, but genomic data are needed to confirm this.

Overall, the present study revealed genome size variation related to the geographic radiation of Haliotis and random changes in species' AT content. Future studies will need to examine the karyotypes and C-values of abalones inhabiting other geographic areas to understand the evolutionary chromosomal relationships within the genus Haliotis. For example, chromosomal study using Fluorescence in situ hybridization of Oceanian species will provide detailed information on the karyotype of these species. Estimation of the C-value of Haliotis iris (Gmelin, 1791) in New Zealand showed its close relationship to the North Pacific group (Bester-van der Merwe et al. 2012), and the determination of the chromosome number of Haliotis tuberculata L. in the Mediterranean Sea (In = 28) suggested this area as the origin of earliest abalones (Colombera & Tagliaferri 1983, Arai & Wilkins 1986, Geiger & Groves 1999), which is important for future phylogenetic/phylogeographic studies.


The authors express their gratitude to Dr. Felix G. Ayson, Dr. Teruo Azuma, and all members of the abalone team in SEAFDEC/AQD in Tigbauan, Iloilo, The Philippines, who assisted in the collection and preparation of abalone samples. The authors are also indebted to Yuki Miyadera and Nobuhiko Ito of the TOSENBO Company Limited and to Suehiro Furukawa, Katsuhiro Furukawa, and Tomoya Ishibasi of the Kitanihon Fishery Company for providing the research materials. This work is supported in part by the JSPS AASP program (2011-2013 fiscal years) awarded to Hokkaido University, Faculty of Fisheries Sciences.


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(1) School of Marine Biosciences, Kitasato University, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan; (2) Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Minato-cho, Hakodate, Hokkaido 041-8611, Japan; (3) Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC/AQD), 5021 Tighauan, Iloilo, Philippines

(*) Corresponding author. E-mail:

DOI: 10.2983/035.037.0518
TABLE 1. Geographic group, diploid chromosome number, C-value, AT
content, FN, and C/n value in Haliotidae.

Geographic group (*)    Species                          2n

European-Mediterranean  Haliotis tuberculata             28 (a.b)
Asian Indo-Pacific      Haliotis asinina                 32 (c)
                        Haliotis varia                   32 (d,c)
                        Haliotis planata                 32 (d)
                        Haliotis diversicolor aquatilis  32 (c)
African Indo-Pacific    Haliotis midae                   36 (f,g)
                        H. midae
Oceanian Indo-Pacific   Haliotis rubra                   36 (h)
                        Haliotis laevigata               36 (h)
                        Haliotis roei                    NA
Pacific Northwest       Haliotis discus hannai           36 (i,j)
                        Haliotis gigantea                36 (k)
                        Haliotis madaka                  36 (l)
Pacific Northeast       Haliotis corrugata               36 (m)
                        Haliotis rufescens               36 (m)
                        Halio tis fulgens                36 (m)

Geographic group (*)    C-value [+ or -] SE (pg)

European-Mediterranean  NA
Asian Indo-Pacific      1.35 [+ or -]0.02
                        1.32 [+ or -]0.04
                        1.52 [+ or -]0.04
                        1.45 (n)
African Indo-Pacific    1.49 [+ or -]0.02
                        1.43 (g)
Oceanian Indo-Pacific   1.93 [+ or -]0.02
                        2.01 [+ or -] 0.04
                        1.76 [+ or -]0.02
Pacific Northwest       1.84 (n)
                        1.73 [+ or -]0.03
                        1.89 [+ or -]0.01
Pacific Northeast       2.14 (m)
                        1.82 (m)
                        1.71 (m)

Geographic group (*)    AT content [+ or -] SE (%)  FN ([dagger])

European-Mediterranean  NA                          56
Asian Indo-Pacific      63.5 [+ or -]0.59           64
                        61.3 [+ or -]0.73           64
                        58.0 [+ or -] 0.60          64
                        66.3 (n)                    62
African Indo-Pacific    63.0 [+ or -]0.38           68

Oceanian Indo-Pacific   65.1 [+ or -]0.47           70
                        66.1 [+ or -]0.45           70
                        64.7 [+ or -] 0.32          NA
Pacific Northwest       62.3 (n)                    72
                        66.0 [+ or -] 0.32          72
                        64.0 [+ or -] 0.28          72
Pacific Northeast       NA                          70
                        NA                          70
                        NA                          68

Geographic group (*)    C/n value (pg)

European-Mediterranean  NA
Asian Indo-Pacific      0.08
African Indo-Pacific    0.08

Oceanian Indo-Pacific   0.11
Pacific Northwest       0.10
Pacific Northeast       0.12

NA, not available.
(*) Geographical grouping described referring to Franchini et al.
(2010) and Geiger and Groves (1999).
([dagger]) Fundamental Number estimated as two for metacentric and
submetacentric chromosome, and one for subtelocentric and telocentric
(a) 'Arai and Wilkins (1986), (b) Colombera and Tagliaferri (1983). (c)
Jarayabhand et al. (1998), (d) Arai et al. (1988), (e) Nakamura (1985),
(f) Van der Merwe and Roodt-Wilding (2008), (e) Franchini et al.
(2010), (h) Botwright (2015), (i) Arai et al. (1982), (j) Okumura et
al. (1999), (k) Miyaki et al. (1997), (l) Miyaki et al. (1999), (m)
Gallardo-Escarate and Del Rio-Portilla (2007), (n) Adachi and Okumura

TABLE 2. Mean C-value of abalones in each geographic group.

Geographic group (*)       N  Mean C-value (pg) SE

Pacific Northeast          3  1.89              0.13
Pacific Northwest          3  1.82              0.05
Oceanian Indo-Pacific      3  1.90              0.07
Asian-African Indo-Pacific 5  1.42              0.04

(*) Geographical grouping described referring to Franchini et al.
(2010) and Geiger and Groves (1999).
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Author:Adachi, Kenta; Arai, Katsutoshi; De La Pena, Milagros R.; Moriyama, Shunsuke; Okumura, Sei-Ichi
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:6SOUT
Date:Dec 1, 2018

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