First extensive examination of genome size in phylum brachiopoda (Lamp Shells) collected from Japan.
KEY WORDS: C value enigma, flow cytometry, brachiopod, lamp shell, Lingula
Genome size (C value) and chromosomal information are fundamental characteristics of every species, and are very important for the progress of cytogenetic, genomic, and phylogenic studies. Within eukaryotes, genome size variations of more than 200,000-fold, from the microsporidium Encephalitozoon cuniculi (0.0029 pg (Biderre et al. 1995)) to the amoeba Amoeba dubia (700 pg (Li 1997), Animal Genome Size Database), have been reported (Gregory 2001, Gregory 2012; Animal Genome Size Database). Because there is no correlation between C value and organismal complexity, this phenomenon was named the C value paradox (Thomas 1971). However, recently, because this paradox is caused by interspecies differences in the amount of noncoding DNA, and C values are not simply correlated to the number of functional genes, it has been recommended that this phenomenon be termed the C value enigma (Gregory 2001). However, these arguments must be developed further because little is known about marine invertebrate C values compared with the number of classification groups and species of these animals. Thus, additional knowledge of invertebrate C values, including that of the less well-known minor phyla, is required.
In the phylum Brachiopoda (lamp shells), which is an important phylum for evolutional studies, the C value of only 2 species have been estimated (Britten & Davidson 1971, Cohen & Gawthrop 1997). Therefore, further studies of C values in brachiopods are necessary to gain insight into C values in the animal kingdom.
The appearance of brachiopod shells is similar to that of bivalves; however, brachiopods are different anatomically from bivalves and are considered to be unrelated evolutionarily to Mollusca (Pennington & Stricker 2001). About 30,000 species and 120 genera of fossil brachiopods have been described in the Lower Cambrian, and today, more than 300 species survive (Rudwick 1970, James et al. 1992, Pennington & Stricker 2001) in all oceans (Cohen & Weydmann 2005). However, there is little cytogenetic knowledge of these living brachiopod species. Only 1 report of chromosomal data is available, for Lingula anatina (Yatsu 1902, Cohen & Weydmann 2005), and C value data are available for only 2 species; these were determined by Hinegardner (unpubl). (Britten & Davidson 1971, Cohen & Gawthrop 1997) for Glottidia pyramidata (0.43 pg, 4.15 x [10.sup.8] bp) and Lingula sp. (0.38 pg, 3.67 x [10.sup.8] bp). Moreover, although Brachiopoda is composed of 2 classes (Inarticulata and Articulata), and 2 orders in each of these classes, the 3 previously mentioned species all belong to the same order (Lingulida) and class (Inarticulata). Therefore, cytogenetic investigations over a wider range of classification within Brachiopoda are needed. In this study, we used flow cytometry to determine the C values of 8 species of brachiopods belonging to 2 classes and 3 orders--collected from Japan.
MATERIALS AND METHODS
The specimens used in this study are summarized in Table 1. The specimens of Lingula anatina (Inarticulata, Lingulida) were collected from Amami Island, Kagoshima, Japan, in May 2012. The specimens of Lingula reevii (Inarticulata, Lingulida), caught in the Ariake Sea, Nagasaki, Japan, were obtained from a fish market in August 2009. A Discradisca stella specimen (Inarticulata, Acrotretida) was collected by dredging at a depth of 42-44 m off Omishima Island (Ehime) in the Inland Sea of Japan in April 2012. Specimens of another 5 species--Terebratulina crossei, Laqueus rubellus, Laqueus blanfordi, Terebratalia coreanica, and Platidia japonica--belonging to articulated brachiopods (Terebratulida) were obtained from fisherman using gill nets at a depth of approximately 100 m in Okirai Bay, Iwate, Japan, in February 2010. Specimens of P. japonica that were attached to the shell surface of T. crossei were also collected. These specimens were dissected, and the lophophore tissue samples were used for C value analysis, as follows.
C Value Analysis
C values were measured using a model PA flow cytometer (Partec Company, Germany), which had been adapted for measurement of the propidium iodide (PI) wavelength (Adachi & Okumura 2012). In our previous study, we found that the C value of the abalone Haliotis discus hannai was 1.84 pg (Adachi & Okumura 2012). Therefore, in this study, the C values of each brachiopod were measured using cephalic tentacle cells of abalone as the internal standard.
The samples were placed in 1.5-mL microcentrifuge tubes with 2 or 3 drops of solution A (CyStain DNA 2-step extraction buffer; Partec), crushed by injection needle, and maintained at room temperature for 5 min. To each sample, we added 700 [micro]L staining buffer (Partec), after which samples were filtered through a 50-[micro]m mesh filter (CellTrics Filter; Partec), before adding 1.5 [micro]L RNase A and 3 [micro]L PI stock solution (Partec). Last, these stained cell suspensions were kept in the dark at 4[degrees]C for 30 min before analysis by flow cytometry. Each sample was analyzed across a minimum of 5,000 nuclei (Adachi & Okumura, 2012). The C values (measured in picogram) and haploid genome sizes (measured in base pairs, converted from the C value) of the various brachiopods were calculated according to published methods (Dolezel et al. 2003, Rees et al. 2008).
A typical flow cytometry histogram of relative fluorescence intensities of PI in the brachiopods and Haliotis discus hannai as standard cells is shown in Figure 1. The determined C values (measured in picogram) and their haploid genome sizes (measured in base pairs) for 8 brachiopods--Lingula anatina, Lingula reevii, Discradisca stella, Terebratulina crossei, Laqueus rubellus, Laqueus blanfordi, Terebratalia coreanica, and Platidia japonica--are shown in Table 1. The C values ranged from 0.31 pg (P. japonica)--0.46 pg (D. stella), showing 1.48-fold variation. When comparing the different classes of brachiopods, we found that C values in inarticulate brachiopods ranged from 0.41 pg in L. anatina--0.46 pg in D. stella (l.12-fold variation); in articulated brachiopods, C values ranged from 0.31 pg in P. japonica--0.44 pg in L. rubellus (1.42-fold variation).
In this study, we determined the C values of 8 brachiopod species collected from Japan, thus increasing the sparse knowledge about this aspect of invertebrate marine animals.
C value variation of marine vertebrates has been reported to range from 0.35 pg in puffer fish (Tetraodon fluviatilis (Lamatsch et al. 2000))--142 pg in the lungfish Protopterus aethiopicus (Pedersen 1971, Hickey & Clements 2005). In major marine invertebrate groups, C value variations have been reported as 125-fold in nematodes, 340-fold in flatworms, 130-fold in annelids, 18-fold in molluscs, 460-fold in crustaceans, and 8-fold in echinoderms (Gregory 2012; Animal Genome Size Database).
As described earlier, Brachiopoda has been subdivided into the 2 classes Inarticulata and Articulata. Inarticulata comprises 2 orders (Lingulida and Acrotretida): Articulata comprises an additional 2 orders (Rhynchonellida and Terebratulida). Although we investigated the C values of species belonging to 3 of the 4 orders (Rhynchonellida was the exception), the variation in C values in these 3 orders of brachiopods was only 1.48-fold. When previous data on 2 other species (Britten & Davidson 1971, Cohen & Gawthrop 1997) were added to our results, this variation range did not change. Moreover, there was not much difference in the C values between each of the 2 orders within the classes of Inarticulata and Articulata when including the previous data (Britten & Davidson 1971, Cohen & Gawthrop 1997). These findings suggested that the C value of brachiopods is very low, and that the range of C value variation in brachiopods is markedly lower than that in other animal phyla. The C value of Platidia japonica, which belongs to suborder Terebratelidina, showed the lowest C value among the 10 species of brachiopods (the current study and Hinegardner's study, as cited by Britten & Davidson (1971) and Cohen & Gawthrop (1997)). In the future, additional species of Terebratelidina should be examined.
The C value data were compared with those of the phylum Bryozoa, which is related closely to the phylum Brachiopoda. The C values in bryozoans have been reported for 26 species (Gregory 2012; Animal Genome Size Database). The C value variation of bryozoans was reported to be 8-fold, ranging from 0.20 pg in Bugula sp. (Pisano 1991) and Watersipora sp. (Schopf 1985) to 1.60 pg in Beania magellanica (Pisano 1991, Gregory 2012: Animal Genonae Size Database). Therefore, even when comparing closely related phyla, the C value variation in brachiopods is less.
It is accepted that C value changes are caused by changes in the number of transposable elements, in intron sizes, polyploidy, and DNA loss (Gregory 2005). This low C value variation suggests that fewer evolutional events occurred in brachiopods than in other taxa of the animal kingdom.
In future studies, the C values of species from the order Rhynchonellida, which have not yet been determined, must be examined, and relationships between C values and phyletic evolution should be examined because the phyletic relationship of brachiopods has not yet been clarified. The brachiopods C values revealed in this study will contribute to the genome sequence and cytogenetic examination for phyletic studies in Brachiopoda.
We are indebted to Matsuya Kawahata of Koushinmaru for generously donating research materials. We are grateful to Suehiro Furukawa and Tomoya Ishibasi of the Kitanihon Fishery Company for their contribution of research materials.
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KENTA ADACHI, (1) TAKASHI KURAMOCHI, (2) KAZUMA KIMURA (1) AND SEI-ICHI OKUMURA (1), *
(1) Kitasato University, School of Marine Biosciences, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan; (2) Hayama shiosai Museum, Miura-gun, Hayama, Kanagawa 240-0111, Japan
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Taxonomic positions, collection areas, and C values in brachiopods. Collected Taxa Species Locality n Class Inarticulata Order Lingulida Family Lingulidae Lingula anatina Amami Island 7 Lingula reevii Ariake Sea 10 Lingula sp. NA NA Glottidia pyramidata NA NA Order Acrotretida Family Discinidae Discradisca stella Inland Sea 1 Class Articulata Order Terebratulida Suborder Terebratelidina Family Terebratulida crossei Okirai Bay 10 Cancellothyrididae Family Laqueidae Laqueus rubellus Okirai Bay 5 Laqueus blanfordi Okirai Bay 3 Terebratalia Okirai Bay 1 coreanica Suborder Terebratelidina Family Platidiidae Platidia japonica Okirai Bay 3 Haploid DNA content C value Taxa Species [+ or -] SE (pg) Class Inarticulata Order Lingulida Family Lingulidae Lingula anatina 0.41 [+ or -] 0.01 Lingula reevii 0.41 [+ or -] 0.003 Lingula sp. 0.38 Glottidia pyramidata 0.43 Order Acrotretida Family Discinidae Discradisca stella 0.46 Class Articulata Order Terebratulida Suborder Terebratelidina Family Terebratulida crossei 0.33 [+ or -] 0.01 Cancellothyrididae Family Laqueidae Laqueus rubellus 0.44 [+ or -] 0.02 Laqueus blanfordi 0.42 [+ or -] 0.01 Terebratalia 0.37 coreanica Suborder Terebratelidina Family Platidiidae Platidia japonica 0.31 [+ or -] 0.005 Haploid DNA content Genome size Taxa Species [+ or -] SE (Mbp) Class Inarticulata Order Lingulida Family Lingulidae Lingula anatina 403.61 [+ or -] 9.52 Lingula reevii 401.29 [+ or -] 2.61 Lingula sp. 367.00 Glottidia pyramidata 415.00 Order Acrotretida Family Discinidae Discradisca stella 449.88 Class Articulata Order Terebratulida Suborder Terebratelidina Family Terebratulida crossei 323.91 [+ or -] 11.66 Cancellothyrididae Family Laqueidae Laqueus rubellus 428.29 [+ or -] 18.63 Laqueus blanfordi 407.89 [+ or -] 4.90 Terebratalia 359.9 coreanica Suborder Terebratelidina Family Platidiidae Platidia japonica 299.92 [+ or -] 4.90 Taxa Species Reference Class Inarticulata Order Lingulida Family Lingulidae Lingula anatina Current study Lingula reevii Current study Lingula sp. Britten and Davidson (1971), Cohen and Gawthrop (1997) Glottidia pyramidata Britten and Davidson (1971), Cohen and Gawthrop (1997) Order Acrotretida Family Discinidae Discradisca stella Current study Class Articulata Order Terebratulida Suborder Terebratelidina Family Terebratulida crossei Current study Cancellothyrididae Family Laqueidae Laqueus rubellus Current study Laqueus blanfordi Current study Terebratalia Current study coreanica Suborder Terebratelidina Family Platidiidae Platidia japonica Current study NA, not available.
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|Author:||Adachi, Kenta; Kuramochi, Takashi; Kimura, Kazuma; Okumura, Sei-ichi|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2013|
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