Research Article |
Corresponding author: Xiao-dong Zheng ( xdzheng@ouc.edu.cn ) Academic editor: Valeria Specchia
© 2017 Jin-hai Wang, Xiao-dong Zheng.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wang J-h, Zheng X-d (2017) Comparison of the genetic relationship between nine Cephalopod species based on cluster analysis of karyotype evolutionary distance. Comparative Cytogenetics 11(3): 477-494. https://doi.org/10.3897/compcytogen.v11i3.12752
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Karyotype analysis was carried out on gill cells of three species of octopods using a conventional air-drying method. The karyotype results showed that all the three species have the same diploid chromosome number, 2n=60, but with different karyograms as 2n=38M+6SM+8ST+8T, FN (fundamental number)=104 (Cistopus chinensis Zheng et al., 2012), 2n=42M+6SM+4ST+8T, FN=108 (Octopus minor (Sasaki, 1920)) and 2n=32M+16SM+12T, FN=108 (Amphioctopus fangsiao (d’Orbigny, 1839–1841)). These findings were combined with data from earlier studies to infer the genetic relationships between nine species via cluster analysis using the karyotype evolutionary distance (De) and resemblance-near coefficient (λ). The resulting tree revealed a clear distinction between different families and orders which was substantially consistent with molecular phylogenies. The smallest intraspecific evolutionary distance (De=0.2013, 0.2399) and largest resemblance-near coefficient (λ=0.8184, 0.7871) appeared between O. minor and C. chinensis, and Sepia esculenta Hoyle, 1885 and S. lycidas Gray, 1849, respectively, indicating that these species have the closest relationship. The largest evolutionary gap appeared between species with complicated karyotypes and species with simple karyotypes. Cluster analysis of De and λ provides information to supplement traditional taxonomy and molecular systematics, and it would serve as an important auxiliary for routine phylogenetic study.
octopods, cytogenetics, chromosome, genetic relationship, evolutionary distance
Cephalopoda is an old and evolutionarily successful molluscan group with a worldwide distribution (
Karyotype analysis is the foundation of cytogenetic studies, playing an important role in understanding the origin and evolution of organisms by studying the variation in the number or structure of their chromosomes (
Species | Origin | Karyotype | References | |||
Locations | Materials | 2n | FN | Formulas | ||
O. minor | Weihai, Shandong Province, China | gills | 60 | 108 | 42M+6SM+4ST+8T | This study |
O. vulgaris | Nagasaki, Japan | embryos | 60 | 76 | 14M+2SM+8ST+36T |
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A. fangsiao | Qingdao, Shandong Province, China | gills | 60 | 108 | 32M+16SM+12T | This study |
C .chinensis | Ningde, Fujian Province, China | gills | 60 | 104 | 38M+6SM+8ST+8T | This study |
S. lycidas | Ohmura, Nagasaki, Japan | wild eggs | 92 | 172 | 66M+14SM+10ST+2T |
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S. esculenta | Shimabara, Nagasaki, Japan | wild eggs | 92 | 164 | 48M+24SM+14ST+6T |
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S. lessoniana | Nomozaki, Nagasaki, Japan | wild eggs | 92 | 156 | 54M+10SM+24ST+4T |
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P. edulis | Nagasaki, Japan | embryos | 92 | 160 | 50M+18SM+16ST+8T |
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H. bleekeri | Nagasaki, Japan | embryos | 92 | 166 | 54M+20SM+18ST |
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Karyotype evolutionary distance has been used as an important parameter in studying the classification and evolution of animals. In this approach, the distance of karyotype evolution (De) and resemblance-near coefficients (λ) are estimated from the karyotype data by mathematical statistics based on the principles of numerical taxonomy and similar analysis theory, and these parameters accurately reflect the interspecific or intraspecific relationship at the cytological level. While the classification and genetic relationships of cephalopods is a continuing topic of interest and has been addressed using molecular systematics tools, such as mitochondrial DNA (
Here, we use a cytogenetic approach to study the genetic relationships of cephalopods at the chromosome level. We used gills to obtain good metaphase mitotic plates, and then calculated the De and λ in order to construct a cluster analysis diagram among nine species cephalopods. These findings enrich our knowledge of cephalopod chromosome structure and provide a new and important index for cephalopod taxonomic classification and the determination of genetic relationships at the cytological level.
We obtained ten live O. minor specimens from the Rongcheng coastal waters of the Bohai Sea (37°13'N, 122°33'E), Shandong Province, China, and ten specimens of A. fangsiao were from the Qingdao coastal waters of the Yellow Sea (36°06'N, 120°32'E), Shandong Province, China. Another ten C. chinensis was transported to laboratory in plastic bags with oxygenation, at a low temperature, from the Ningde coastal waters of the East Sea (27°18'N, 119°32'E), Fujian Province, China. All individuals were about 40g and were identified based on morphological characteristics.
Chromosome preparation followed the method of
Microphotographs of the chromosomes were used for karyotype analysis with Image-Pro Plus 6.0 (
We used the chromosome relative length as karyotype parameter of nine species (three from this study) for the analysis of evolutionary relationships (Table
Karyological analysis of Giemsa-stained chromosomes was successfully obtained from at least seven well- divided metaphase plates from the studied populations of O. minor, A. fangsiao and C. chinensis (Fig.
Photomicrographs of somatic diploid metaphase plates and karyotypes from three species of octopod gills. A The metaphase plate of O. minor B Karyogram of O. minor from (A) showing the karyotype composition: 42 metacentric (#1–#21), 6 submetacentric (#22–#24), 4 subtelocentric (#25–#26), and 8 telocentric (#27–#30) chromosomes C The metaphase plate of A. fangsiao D Karyogram of A. fangsiao from (C) showing the karyotype composition: 32 metacentric (#1–#16), 16 submetacentric (#17–#24), and 12 telocentric (#25–#30) chromosomes E The metaphase plate of C. chinensis F Karyogram of C. chinensis from (E) showing the karyotype composition: 38 metacentric (#1–#19), 6 submetacentric (#20–#22), 8 subtelocentric (#23–#26), and 8 telocentric (#27–#30) chromosomes. Scale bar 5 μm.
Comparison of karyotype parameters obtained among O. minor, A. fangsiao and C. chinensis. (SA, short arm relative length; LA, long arm relative length; AR, arm ratio=LA/SA; CI, centromeric index=SA/(SA+LA) ×100; M, metacentric, 1.0 < AR < 1.7; SM, submetacentric, 1.7 < AR < 3.0; ST, subtelocentric, 3.0 < AR < 7.0; T, telocentric, 7.0 < AR. Values as mean ± SE)
Chromosome no. | Octopus minor (42M+6SM+4ST+8T) | Amphioctopus fangsiao (32M+16SM+12T) | Cistopus chinensis (38M+6SM+8ST+8T) | |||||||||||||||
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SA | LA | SA+LA | AR | CI | Type | SA | LA | SA+LA | AR | CI | Type | SA | LA | SA+LA | AR | CI | Type | |
1 | 2.43 | 2.54 | 4.97±0.17 | 1.05±0.15 | 48.89 | M | 3.33 | 3.55 | 6.88±0.18 | 1.06±0.08 | 48.46 | M | 4.13 | 4.15 | 8.28±0.05 | 1.00±0.10 | 49.88 | M |
2 | 2.28 | 2.49 | 4.77±0.11 | 1.09±0.12 | 47.80 | M | 2.99 | 3.46 | 6.45±0.70 | 1.16±0.12 | 46.36 | M | 3.64 | 3.75 | 7.39±0.11 | 1.03±0.02 | 49.26 | M |
3 | 2.22 | 2.51 | 4.73±0.05 | 1.13±0.08 | 46.93 | M | 3.04 | 3.15 | 6.19±0.47 | 1.03±0.02 | 49.17 | M | 3.60 | 3.78 | 7.38±0.07 | 1.05±0.02 | 48.78 | M |
4 | 2.30 | 2.43 | 4.73±0.13 | 1.06±0.12 | 48.63 | M | 2.86 | 2.87 | 5.73±0.19 | 1.00±0.11 | 49.91 | M | 3.15 | 3.44 | 6.59±0.12 | 1.09±0.03 | 47.80 | M |
5 | 2.36 | 2.36 | 4.72±0.06 | 1.00±0.13 | 50.00 | M | 2.53 | 2.96 | 5.49±0.07 | 1.17±0.17 | 46.01 | M | 3.33 | 3.35 | 6.68±0.09 | 1.01±0.11 | 49.85 | M |
6 | 2.28 | 2.36 | 4.64±0.01 | 1.04±0.05 | 49.14 | M | 1.94 | 2.12 | 4.06±0.20 | 1.09±0.04 | 47.78 | M | 3.14 | 3.18 | 6.32±0.15 | 1.01±0.04 | 49.68 | M |
7 | 2.21 | 2.38 | 4.59±0.05 | 1.08±0.01 | 48.15 | M | 1.97 | 2.09 | 4.06±0.10 | 1.06±0.01 | 48.52 | M | 2.97 | 3.19 | 6.16±0.04 | 1.07±0.01 | 48.21 | M |
8 | 2.19 | 2.40 | 4.59±0.11 | 1.10±0.14 | 47.71 | M | 1.66 | 2.18 | 3.84±0.21 | 1.31±0.01 | 43.23 | M | 2.83 | 2.98 | 5.81±0.20 | 1.05±0.07 | 48.71 | M |
9 | 2.16 | 2.38 | 4.54±0.05 | 1.12±0.02 | 47.58 | M | 1.82 | 1.91 | 3.73±0.22 | 1.05±0.02 | 48.79 | M | 2.42 | 2.48 | 4.91±0.17 | 1.02±0.11 | 49.29 | M |
10 | 2.13 | 2.15 | 4.28±0.12 | 1.01±0.10 | 49.78 | M | 1.63 | 2.07 | 3.70±0.06 | 1.27±0.01 | 44.05 | M | 2.29 | 2.44 | 4.73±0.06 | 1.07±0.06 | 48.41 | M |
11 | 2.12 | 2.16 | 4.28±0.03 | 1.01±0.07 | 49.53 | M | 1.55 | 1.95 | 3.50±0.31 | 1.26±0.08 | 44.29 | M | 2.28 | 2.45 | 4.73±0.15 | 1.07±0.08 | 48.20 | M |
12 | 2.03 | 2.17 | 4.20±0.14 | 1.07±0.13 | 48.33 | M | 1.68 | 1.76 | 3.44±0.11 | 1.05±0.04 | 48.84 | M | 2.24 | 2.27 | 4.51±0.11 | 1.01±0.03 | 49.67 | M |
13 | 1.92 | 2.04 | 3.96±0.07 | 1.06±0.09 | 48.48 | M | 1.42 | 1.79 | 3.21±0.27 | 1.26±0.21 | 44.24 | M | 1.99 | 2.31 | 4.40±0.07 | 1.16±0.01 | 45.23 | M |
14 | 1.91 | 1.98 | 3.89±0.15 | 1.04±0.14 | 49.10 | M | 1.56 | 1.64 | 3.20±0.04 | 1.05±0.13 | 48.75 | M | 1.96 | 2.27 | 4.23±0.10 | 1.16±0.03 | 46.34 | M |
15 | 1.88 | 1.98 | 3.86±0.09 | 1.05±0.10 | 48.70 | M | 1.47 | 1.69 | 3.16±0.32 | 1.15±0.32 | 46.52 | M | 2.03 | 2.10 | 4.13±0.16 | 1.03±0.12 | 49.15 | M |
16 | 1.36 | 1.90 | 3.26±0.11 | 1.40±0.13 | 41.72 | M | 1.32 | 1.69 | 3.01±0.05 | 1.28±0.13 | 43.85 | M | 1.75 | 1.99 | 3.74±0.05 | 1.14±0.07 | 46.79 | M |
17 | 1.42 | 1.54 | 2.96±0.12 | 1.08±0.18 | 47.97 | M | 2.48 | 4.39 | 6.87±0.13 | 1.77±0.28 | 36.10 | SM | 1.58 | 1.96 | 3.54±0.03 | 1.24±0.08 | 44.63 | M |
18 | 1.28 | 1.29 | 2.57±0.05 | 1.01±0.10 | 49.81 | M | 2.37 | 4.30 | 6.67±0.07 | 1.81±0.14 | 35.53 | SM | 1.55 | 1.55 | 3.10±0.02 | 1.00±0.04 | 50.00 | M |
19 | 1.24 | 1.27 | 2.51±0.13 | 1.02±0.11 | 49.40 | M | 1.92 | 3.59 | 5.51±0.24 | 1.87±0.29 | 34.85 | SM | 1.02 | 1.23 | 2.25±0.04 | 1.21±0.06 | 45.33 | M |
20 | 0.93 | 1.19 | 2.12±0.08 | 1.28±0.10 | 43.87 | M | 2.02 | 3.46 | 5.48±0.09 | 1.71±0.12 | 36.86 | SM | 1.52 | 4.53 | 6.05±0.08 | 2.98±0.06 | 25.12 | SM |
21 | 0.95 | 0.96 | 1.91±0.10 | 1.01±0.05 | 49.74 | M | 1.53 | 2.66 | 4.19±0.26 | 1.74±0.20 | 36.52 | SM | 1.23 | 2.46 | 3.69±0.16 | 2.00±0.20 | 33.33 | SM |
22 | 1.85 | 3.14 | 4.99±0.05 | 1.70±0.05 | 37.07 | SM | 1.12 | 2.50 | 3.62±0.18 | 2.23±0.11 | 30.94 | SM | 1.02 | 2.42 | 3.44±0.08 | 2.37±0.08 | 29.65 | SM |
23 | 1.01 | 2.87 | 3.88±0.10 | 2.84±0.11 | 35.19 | SM | 1.05 | 2.53 | 3.58±0.19 | 2.41±0.01 | 29.33 | SM | 1.15 | 4.63 | 5.78±0.09 | 4.03±0.04 | 19.90 | ST |
24 | 0.97 | 2.90 | 3.87±0.09 | 2.99±0.02 | 25.06 | SM | 0.74 | 2.13 | 2.87±0.03 | 2.88±0.30 | 25.78 | SM | 1.10 | 3.89 | 4.99±0.03 | 3.54±0.01 | 22.04 | ST |
25 | 1.03 | 3.52 | 4.55±0.13 | 3.42±0.13 | 22.64 | ST | 0.32 | 2.86 | 3.18±0.66 | 8.90±0.13 | 10.10 | T | 0.55 | 3.35 | 3.90±0.16 | 6.09±0.13 | 14.10 | ST |
26 | 0.83 | 3.17 | 4.00±0.01 | 3.82±0.04 | 20.75 | ST | 0.22 | 2.88 | 3.10±0.40 | 12.90±0.32 | 7.20 | T | 0.73 | 3.15 | 3.88±0.10 | 4.32±0.13 | 18.81 | ST |
27 | - | 3.02 | 3.02±0.01 | ∞ | - | T | - | 2.71 | 2.71±0.42 | ∞ | - | T | - | 2.71 | 2.71±0.02 | ∞ | - | T |
28 | - | 3.02 | 3.02±0.05 | ∞ | - | T | - | 2.54 | 2.54±0.16 | ∞ | - | T | - | 2.74 | 2.74±0.06 | ∞ | - | T |
29 | - | 1.77 | 1.77±0.01 | ∞ | - | T | - | 1.90 | 1.90±0.03 | ∞ | - | T | - | 1.69 | 1.69±0.07 | ∞ | - | T |
30 | - | 1.15 | 1.15±0.07 | ∞ | - | T | - | 0.90 | 0.90±0.03 | ∞ | - | T | - | 1.56 | 1.56±0.10 | ∞ | - | T |
We compared the relative chromosome length of the nine species of cephalopods and plotted a detailed chromosome distribution diagram to show the number and proportion of the different types of chromosome in the different species (Fig.
We developed a novel method to create normative karyo-idiograms of the three species based on the karyotype parameters (Fig.
Karyotypes vary greatly between species, with greater karyotype evolutionary distance (De) and smaller resemblance-near coefficients (λ) between distantly related species. Likewise, the karyotype evolutionary distance within a family is generally smaller than that between different families. To make an integrative analysis of the genetic relationships, the De and λ values of the nine cephalopods were calculated (Table
The karyotype evolutionary distance and resemblance-near coefficient among nine species of cephalopods.
Species | O. minor | O. vulgaris | A. fangsiao | C. chinensis | S. lycidas | S. esculenta | S. lessoniana | P. edulis | H. bleekeri |
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O. minor | 0.5894 | 0.7594 | 0.8184 | 0.5244 | 0.3747 | 0.4401 | 0.4839 | 0.2742 | |
O. vulgaris | 0.5291 | 0.5495 | 0.5540 | 0.4183 | 0.3765 | 0.4725 | 0.4392 | 0.3057 | |
A. fangsiao | 0.2760 | 0.6000 | 0.7976 | 0.5423 | 0.5075 | 0.4515 | 0.4963 | 0.2640 | |
C. chinensis | 0.2013 | 0.5912 | 0.2262 | 0.5343 | 0.4744 | 0.5467 | 0.3846 | 0.3663 | |
S. lycidas | 0.6460 | 0.8722 | 0.6122 | 0.6271 | 0.7871 | 0.6328 | 0.6297 | 0.5990 | |
S. esculenta | 0.9809 | 0.9782 | 0.6776 | 0.7471 | 0.2399 | 0.5809 | 0.5594 | 0.6051 | |
S. lessoniana | 0.8214 | 0.7494 | 0.7960 | 0.6034 | 0.4570 | 0.5431 | 0.5280 | 0.3650 | |
P. edulis | 0.7265 | 0.8230 | 0.7011 | 0.9550 | 0.4620 | 0.5822 | 0.5904 | 0.5984 | |
H. bleekeri | 1.2954 | 1.1845 | 1.3323 | 1.0101 | 0.5120 | 0.5030 | 0.6940 | 0.5140 |
In order to shed further light on phylogenetic divergence within the clades Octopoda, Sepiida and Teuthida, a cluster analysis was applied (Fig.
A Relationships between chromosome number and UPGMA clustering of nine species of cephalopods by evolutionary distance with simplified karyo-idiogram and karyotype formulas. Chromosome numbers and De values are shown on the corresponding branches B Phylogenetic relationships among the cephalopods based on mitochondrial DNA sequences including the nine species of this study (
In previous reports, germ cells, blood cells, and embryos (
The chromosome number of the three species in the present study was 2n=60, which is consistent with previous karyotype studies of octopods (
Despite the three octopods having the same number of chromosomes, the karyotypes were remarkably different from each other. Compared with O. minor and C. chinensis, A. fangsiao had a specialized karyotype without ST, while the former two had almost the same karyotype, with only slight differences in M and ST (Fig.
Chromosome distribution diagram of nine species of cephalopods. The slopes of the four lines are 1, 1.7, 3 and 7, dividing the diagram into four zones which represent four types of chromosome. SA, short arm relative length; LA, long arm relative length; M, metacentric; SM, submetacentric; ST, subtelocentric; T, telocentric. The blue area of pie charts and bar charts means M+SM and M, respectively.
Earlier analyses of cephalopod genetic relationships mainly concentrated on phylogenetic reconstruction via specific rDNA sequences (
In view of this, more detailed cephalopod chromosome information is urgently needed to facilitate comprehensive analyses of genetic relationships at the cytological level. Fluorescence in situ hybridization (FISH), which enables visualization of target DNA sites on chromosomes through a signal display using probes, has been widely applied in chromosomal localization (
In this study, we revealed the karyotypes of three octopods, bringing the total to nine reliable cephalopod karyotypes. Furthermore, this is the first study to determine the genetic relationship among these nine species at the cytological level by cluster analysis based on the karyotype evolutionary distance and resemblance-near coefficient. Our results demonstrated the feasibility of De cluster analysis for cephalopod taxonomic classification, which could serve an important auxiliary means of routine phylogenetic analysis and provide insights into chromosome evolution.
We also thank YS Qian, QZ Ke and B Cai for providing the O. minor and C. chinensis individuals from Rongcheng and Ningde, China. This study was supported by research grants from National Natural Science Foundation of China (No31672257) and Key Development Plan of Shandong Province (2016GSF115014).
Chromosome relative length, supplemental formulae
Data type: statistical data
Explanation note: Chromosome relative length, supplemental formulae and all of the original images are made available under the online digital repository Figshare, and it is free to access, in adherence to the principle of open data, more details in https://figshare.com/s/8d21a0db9ffe1f17d279