gene duplications and the evolution of c-type lysozyme...

ADVANCES IN CICHLID RESEARCH II

Gene duplications and the evolution of c-type lysozymeduring adaptive radiation of East African cichlid fish

Shiho Takahashi-Kariyazono .

Hirokazu Tanaka . Yohey Terai

Received: 22 February 2016 / Revised: 14 June 2016 / Accepted: 18 June 2016 / Published online: 1 July 2016

� Springer International Publishing Switzerland 2016

Abstract The adaptive radiation of African cichlids

is a prime model system for studying vertebrate

speciation. Cichlid species are distributed in lakes and

rivers with various water conditions such as various

pH values. The innate immune system in fish is

particularly important because water contains a wide

range of pathogenic microorganisms. To investigate

the evolution of the host defense system in cichlids, we

isolated the c-type lysozyme gene, which functions in

the innate immune system of fish. Southern blot and

sequence analyses showed that the lysozyme gene

underwent several gene duplication events and

evolved with amino acid replacements during the

adaptive radiation of cichlids. The inferred 3D struc-

ture revealed that the amino acid substitutions were

localized on the lysozyme surface. Moreover, more

than half of the surface substitutions changed the

charge of amino acid residues, suggesting changes in

the optimum pH for enzymatic activity. In African

cichlids, the lysozyme genes may have played and still

play an important role in defense against pathogens.

Keywords c-Type lysozyme � Gene duplication �Adaptation � East African cichlids

Introduction

Gene duplication is a major source of genetic variation

and can lead to the generation of functional genes

(Ohno, 1970; Zhang, 2003; Conant & Wolfe, 2008;

Makino & Kawata, 2012). One copy of a duplicated

gene retains the ancestral function and the other copy

is freed from purifying selection and may acquire a

new function (Zhang, 2003; Conant & Wolfe, 2008).

The evolution of such redundant copies has con-

tributed to adaptation to environmental variation by

enabling the acquisition of new gene functions (Kon-

drashov, 2012). Indeed, the proportion of duplicated

genes is related to environmental diversity in Droso-

phila species, indicating that variation due to gene

Shiho Takahashi-Kariyazono and Yohey Terai have

contributed equally to this work.

Guest editors: S. Koblmuller, R. C. Albertson, M. J. Genner,

K. M. Sefc & T. Takahashi / Advances in Cichlid Research II:

Behavior, Ecology and Evolutionary Biology

S. Takahashi-Kariyazono � Y. Terai (&)

Department of Evolutionary Studies of Biosystems,

SOKENDAI (The Graduate University for Advanced

Studies), Shonan Village, Hayama, Japan

e-mail: [email protected]

H. Tanaka

Department of Biology and Geosciences, Graduate School

of Science, Osaka City University, 3-3-138, Sugimoto,

Sumiyoshi-ku, Osaka 558-8585, Japan

Y. Terai

Graduate School of Bioscience and Biotechnology, Tokyo

Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,

Yokohama 226-8501, Japan

123

Hydrobiologia (2017) 791:7–20

DOI 10.1007/s10750-016-2892-6

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duplication might be a determinant of the limits of

range expansions (Makino & Kawata, 2012).

Lakes Victoria, Malawi, and Tanganyika in the East

African Rift Valley harbor roughly 700, 700, and 250

endemic species of cichlid fish, respectively (Turner

et al., 2001; Koblmuller et al., 2008b). These fish have

fascinated evolutionary biologists as an example of a

vertebrate adaptive radiation (Fryer & Iles, 1972;

Kocher, 2004). LakeMalawi (with an estimated age of

2–5 million years) (Delvaux, 1995) and Lake Victoria

(with an estimated age of 250,000–750,000 years)

(Johnson et al., 1996) are younger than the oldest Lake

Tanganyika (with an estimated age of 9–12 million

years) (Cohen et al., 1993, 1997). Cichlid species are

distributed in lakes and rivers with pH values ranging

from 6.0 to 10.0 (Fryer & Iles, 1972) in Africa.

Molecular analyses have revealed the phylogenetic

relationships among the major lineages of African

cichlids (Salzburger et al., 2005; Takahashi &

Koblmuller, 2011; Friedman et al., 2013; Weiss

et al., 2015; Takahashi & Sota, 2016). Reflecting the

relative age of the lake, the cichlid lineage of Lake

Tanganyika is the oldest and is derived from riverine

species (Mayer et al., 1998; Terai et al., 2003;

Takahashi & Sota, 2016). A few Lake Tanganyika

species reinvaded rivers and their progenies indepen-

dently colonized Lakes Malawi and Victoria (Sal-

zburger et al., 2005). The fish in each lake underwent

several independent radiations after colonization

(Salzburger et al., 2005; Genner et al., 2007;

Koblmuller et al., 2008b). It is not clear why some

lineages successfully reinvaded riverine habitats and

expanded their distribution. A genomic study of five

African cichlids revealed an excess of gene duplica-

tions in the East African lineage (Brawand et al.,

2014), suggesting the importance of gene duplication

events during their radiation.

Lysozymes are hydrolytic enzymes that cleave the

peptidoglycan in bacterial cell walls (Callewaert &

Michiels, 2010). Three types of lysozymes have been

identified in the animal kingdom, c-type (chicken),

g-type (goose type), and i-type (invertebrate type)

(Callewaert & Michiels, 2010). The enzymatic activ-

ity of lysozymes is widely used for antibacterial host

defense and digestion (Dobson et al., 1984; Jolles

et al., 1996). In fish, lysozyme functions in the innate

immune system and is considered to be the first line of

defense against various bacteria (Saurabh & Sahoo,

2008). The fish innate immune system is particularly

important because water contains a wide range of

pathogenic microorganisms (Hikima et al., 2001).

Transgenic zebrafish expressing the chicken lysozyme

gene show resistance to pathogenic bacterial infec-

tions, indicating the role of the innate immune system

in defense against bacteria (Yazawa et al., 2006).

Cichlid lysozyme activity was detected from gill,

serum, liver, plasma, skin, and mucus samples of

Oreochromis niloticus Linnaeus, 1758 (On; Sankaran

& Gurnani, 1972; Taoka et al., 2006; Welker et al.,

2007). Considering the important role of the innate

immune system in fish, genetic variation in the

lysozyme gene may be important in the process of

their radiation, especially when they have colonized in

new water environments during the radiation in East

African lakes and rivers. However, the lysozyme gene

has not been analyzed in cichlids from the Great

Lakes.

In this study, we determined the c-type lysozyme

gene from African riverine and lake cichlids. Lyso-

zyme genes have undergone gene duplications during

the adaptive radiation of African cichlids, which may

have contributed to their current distribution in various

water conditions.

Materials and methods

Specimens

Three riverine species, ten Lake Tanganyika species,

six Lake Malawi species, and three Lake Victoria

species were used for analyses (Table 1). All of these

fish were obtained from a commercial source. The

animal protocols and procedures were approved by the

Institutional Animal Care and Use Committees of the

Tokyo Institute of Technology and SOKENDAI. All

surgery was performed under anesthesia, and all

efforts were made to minimize suffering.

DNA and RNA extraction

Genomic DNA was extracted from caudal fin or

muscular tissues using DNeasy blood and tissue kit

(Qiagen, Hilden, Germany). The whole body of fish

was frozen in liquid nitrogen, and then fragmented to

small size. Total RNA was extracted from fragmented

whole body using the RNeasy mini kit (Qiagen).

8 Hydrobiologia (2017) 791:7–20

123

Table 1 Species and detected haplotypes

Species names IDs Determined haplotypes from cDNA (expressed

haplotypes)

Number of bands in

Southern blot

Oreochromis niloticus O. niloticus#1 On1 On2 –

O. niloticus#2 – 2

Steatocranus casuarius S. casuarius#1 Sc1 –

Tilapia buttikoferi T. buttikoferi#1 Tb1 –

Neolamprologus brichardi N. brichardi#1 Nb1 –

Neolamprologus leleupi N. leleupi#1 Nl1 –

Altolamprologus calvus A. calvus#1 Ac1 –

A. calvus#2 – 1

Variabilichromis moorii V. moorii#1 Vm1 –

Lamprologus ocellatus L. ocellatus#1 Lo1 –

Cyphotilapia frontosa C. frontosa#1 Cf1 –

Xenotilapia ochrogenys X. ochrogenys#1 Xo1 –

Cyprichromis leptosoma C. leptosoma#1 Cl1 Cl2 –

C. leptosoma#2 – 1

Spathodus erythrodon S. erythrodon#1 Se1 Se2 –

S. erythrodon#2 – 1

Tropheus duboisi T. duboisi#1 Td1 –

T. duboisi#2 – 1

Aulonocara sp. Aulonocara sp.#1 Au1 Au2 –

Pseudotropheus lombardoi P. lombardoi#1 Pl1 –

P. lombardoi#2 Pl2 –

Pseudotropheus sp. Pseudotropheus sp.#1 Ps1 Ps2 –

Labidochromis caeruleus L. caeruleus#1 Lc2 –

L. caeruleus#2 Lc1 –

L. caeruleus#3 Lc1 Lc3 –

L. caeruleus#4 – 4

Dimidiochromis compressiceps D. compressiceps#1 Dc1 Dc5 –

D. compressiceps#2 Dc6, and four other partial haplotypes (Fig. 5) –

D. compressiceps#3 Dc3 Dc4 –

D. compressiceps#4 Dc4 –

D. compressiceps#5 Dc2 Dc4

D. compressiceps#6 Determined from genomic DNA: Dc1 Dc2 Dc3

Dc6 and one other haplotypes (Fig. 5)

4

Melanochromis auratus M. auratus#1 Ma1 –

Haplochromis brownae H. brownae#1 Hb1 Hb2 –

H. nigricans H. nigricans#1 Hn1 Hn2 –

H. sp. ‘redtail sheller’ H. sp. ‘redtail sheller’#1 Hr1 –

H. sp. ‘redtail sheller’#2 – 2

Hydrobiologia (2017) 791:7–20 9

123

Isolation of lysozyme cDNA and gene

cDNAs were synthesized using a cDNA synthesis kit

(Takara, Shiga, Japan). A partial cDNA of cichlid

lysozymewas cloned by RT-PCR using primers LysdF1

(50-TCTTCCAGCGCTGTGAATGG-30) and LysdR1

(50-TATTGATCTGAAAGATGCCATAGTC-30), andfull-length cDNAs were isolated from the RNA of

Labidochromis caeruleus Fryer, 1956 by 30- and 50

RACE using the nested primers LysU1 (50-CAAACGGGATGGATGGCTATCGT-30), LysU2 (50-ACAAAGGCAACAAATCGTAACACTGATG-30), LysD1 (50-CATCAGTGTTACGATTTGTTGCCTTTGT-30), and

LysD2 (50-CTGACACCACGATAGCCATCCATC-30).PCR was performed in a PTC-100 programmable

thermal controller (MJ Research, Waltham, MA, USA)

and the GeneAmp PCR system 9700 (Applied Biosys-

tems, Carlsbad, CA, USA). The PCR program for

isolation of partial cDNAconsisted of a denaturation step

for 3 min at 94�C, followed by 30 cycles of denaturationfor 1 min at 94�C, annealing for 1 min at 55�C, andextension for 1 min at 72�C. The 30- and 50 RACE were

performed with two rounds of nested PCR using LysU1

and LysD1 for the first round, and LysU2 and LysD2 for

the second round. The first and second PCR were

performed under the same conditions consisting of a

denaturation step for 3 min at 94�C, followed by 30

cycles of denaturation for 1 min at 94�C, annealing for

1 min at 65�C, and extension for 1 min at 72�C. Thelysozyme gene was amplified using LysF1 (50-ATCT-GAACCCAGACAGTCACAG-30) and LysR1 (50-GTCACCCAATGTGTTTTCCTT-30) primers and geno-

mic DNA of L. caeruleus as a template. The PCR

program for isolation of the lysozyme gene consisted of a

denaturation step for 3 min at 94�C, followed by 30

cycles of denaturation for 1 min at 94�C, annealing for

1 minat 55�C, andextension for 3 min at 72�C.ThePCR

product was purified and determined by direct sequenc-

ing using the Applied Biosystems automated 3130

sequencer.

Determination of the lysozyme cDNA sequences

The cDNA fragments containing lysozyme coding

region were amplified by PCR using LysF1 and LysR1

primers and the cDNAof all species used in this study as

templates. The positions of primers are described in

Fig. 1. The PCR program for amplification of the

lysozyme cDNA consisted of a denaturation step for

3 min at 94�C, followed by30 cycles of denaturation for1 min at 94�C, annealing for 1 min at 55�C, and

extension for 1 min at 72�C. The PCR product was

purified and determined by direct sequencing. After the

analysis of the sequences, we used the double peaks in

the sequencing result (color waves) as an indicator of

which PCR product contains two or more sequences.

When we found double peaks in the sequencing results,

we cloned the PCR products into the pGEM-T plasmid

vector (Promega, Madison, WI, USA) and determined

the sequences of several clones to obtain haplotype

information. To amplify lysozyme haplotypes without

annealing bias of PCR primers, we designed primers in

the conserved regions among haplotypes from Lake

Malawi species (Fig. 1, Lys_cnsv_F1: 50-GTTTTCTTGCTTTTGATAACTGTG-30, Lys_exon2F1: 50-TTGCCTGACCAAACATGAGTCAAAC-30, and Lys_cnsv_R1: 50-TTGTTSCCTAACGATACGTTTG-30). The

DNA fragment from exons 1 to 3 was amplified using

Lys_cnsv_F1 and Lys_cnsv_R1 from cDNA of Dimid-

iochromis compressicepsBoulenger, 1908 (Dc,D. com-

pressiceps#2) (Table 1).TheDNAfragment fromexons

2 to 3 with intron 2 was amplified using Lys_exon2F1

and Lys_cnsv_R1 from genomic DNA of D. compres-

siceps#6 (Table 1). The PCR program for amplification

exon 1 exon 2 exon 3 exon 4

200 bp

coding region 429 bp

Lysozyme cDNA

intron 1: probe

LysF1

LysR1

Lys_cnsv_F1 Lys_cnsv_R1Lys_exon2F1_F1

LysF1

LysR1

Lysozyme geneFig. 1 Structure of the

cichlid c-type lysozyme.

Exon positions are indicated

by numbers. Intron 1 was

used as a probe for Southern

blots. The positions of the

primers used for

amplification are indicated

by arrows. Scale bar

represents 200 bp


123

of those fragments consisted of a denaturation step for

3 min at 94�C, followed by30 cycles of denaturation for1 min at 94�C, annealing for 1 min at 55�C, and

extension for 30 s at 72�C. The PCR products were

cloned and determined the sequences as described

above. The nucleotide sequences were deposited in

GenBank under accession numbers LC012556–

LC012592 and LC126611–LC126614.

Southern blot analysis

The partial sequence of lysozyme intron 1 (698 bp)

was amplified using LysintF1 (50-CCAAAATAAG-CAGTTCACCATTG-30) and LysintR1 (50-ACTAACTACTTCTCACATGCTGACACT-30) primers and

the genomic DNA of L. caeruleus as a template. The

PCR product was labeled by [a-32P]dCTP (NEN

Research Products, Boston, MA, USA) and used as a

probe. Genomic DNAs (10 lg each) of Cyprichromis

leptosomaBoulenger, 1898 (Cl) and L. caeruleuswere

digested by EcoRI, HindIII, and PstI, and those of H.

sp. ‘redtail sheller,’ Dc, Tropheus duboisi Marlier,

1959, Spathodus erythrodon Boulenger, 1900 (Se),

Altolamprologus calvus Poll, 1978, and On were

digested by EcoRI and PstI. The digested fragments

were subjected to electrophoresis in a 1% agarose gel.

After electrophoresis, DNA fragments were trans-

ferred from gels to GeneScreen Plus Membranes in

0.4 M NaOH and 0.6 M NaCl. Membranes were

neutralized in 0.5 M Tris–HCl (pH 7.0) and 1 M

NaCl, and then dried. Hybridization was performed at

45�C overnight in a solution of 69 SSC (SSC is

0.15 M NaCl and 0.015 M trisodium citrate, pH 7.0),

1% (w/v) sodium dodecyl sulfate (SDS), 59 Den-

hardt’s reagent [l9 Denhardt’s reagent is 0.02% (w/v)

Ficoll 400, 0.02% (w/v) polyvinylpyrrolidone, and

0.02% (w/v) bovine serum albumin], and 100 pg/ml

herring DNA.Washing was performed in 29 SSC plus

1% SDS at 60�C for 60 min. The signals were detected

by BAS-2000 (Fijix, Fuji Film, Tokyo, Japan).

Genetic analysis

The coding sequences of lysozyme cDNA (429 bp)

were aligned using Genetyx (ver. 10) and was

subjected to a phylogenetic analysis with 1,000

bootstrap replications. A neighbor-joining analysis

(Saitou & Nei, 1987) was performed with MEGA 5

(Tamura et al., 2011) using all substitution sites (87

sites). The sequences from O. niloticus (On2) and

Tilapia buttikoferi Hubrecht, 1881 (Tb1) were used as

outgroup sequences.

Positions of substitutions in the lysozyme 3D

structure

The lysozyme amino acid sequences were aligned

using Genetyx (ver. 10), and all variable positions

were identified. The positions of hen egg white

lysozyme corresponding to variable positions of

cichlid lysozyme were identified from an alignment

of both sequences. Those positions in the 3D structure

of hen egg white lysozyme were observed using Cn3D

software version 4.3 (Wang et al., 2000).

Results

Determination of cichlid lysozyme

We isolated the partial lysozyme sequence from cDNA

of L. caeruleus, and the full-length coding region by

50- and 30 RACE. The coding region was 429 bp in

length. We amplify the lysozyme gene from genomic

DNA of L. caeruleus by PCR, and determined the

sequences. We compared the lysozyme gene

sequences determined from cDNA and genomic

DNA. The genomic structure of the lysozyme gene

consisted of four exons (Fig. 1). The coding regionwas

similar to that of Oreochromis aureus Steindachner,

1864 c-type lysozyme mRNA (EU836689) (420/

429 bp identity). To investigate the evolution of the

lysozyme gene in cichlids, we amplified lysozyme

cDNA from ten Lake Tanganyika species, six Lake

Malawi species, and three Lake Victoria species. In

total, we determined 37 different sequences. We

detected 87 variable sites including 64 nonsynony-

mous and 23 synonymous sites (Fig. 2). Substitutions

in 64 nonsynonymous sites correspond to amino acid

replacements in 47 positions, because several pairs of

nonsynonymous substitutions were located in the same

codon (e.g., substitution in sites 49 and 50 in Fig. 2).

More than two lysozyme haplotypes were isolated

within a species of Lake Malawi cichlids, whereas one

or two haplotypes in Lake Tanganyika cichlids

(Table 1).


123

Lysozyme copy number variation in cichlids

To identify copy number variation of the lysozyme

gene, we performed Southern blot analysis using

intron 1 of the gene as a probe (Fig. 1). First, we used

digested genomic DNA from each of one Lakes

Tanganyika and Malawi species with each of three

restriction enzymes (EcoRI, HindIII, and PstI). We

detected only one signal per lane of a Lake Tan-

ganyika species (Fig. 3A, left panel). In a Lake

Malawi species, four bands were observed in the

digested DNA with EcoRI and PstI, whereas large

sizes of three bands were observed in the lane of

HindIII (Fig. 3A, right panel). The digestion of

genomic DNA with HindIII was not appropriate for

Southern blot analysis, because restriction enzyme

sites were not located near the lysozyme gene. We

additionally analyzed the copy number in one riverine

species, three Lake Tanganyika species, one Lake

Malawi species, and one Lake Victoria species using

two restriction enzymes (EcoRI and PstI). As shown in

Fig. 3B, we detected two signals from each lane of a

Lake Victoria

Lake Malawi

Lake Tanganyika

Riverine

Lam

prol

ogin

i

syn./nonsyn.

nucleotide site

11111111111111222222222222222222222222333333333333333333334444444 12345666666677778889900113456778889013334444555557777888999122344556678888899990000122 698990012378967890190279138509362780730694789016783469678089324646897890138902483456645 snnsnnsnnsnnsnnsnnsnsnnnsnsnnnnnnnnnssnnnnnnsnnnnssnssnnsnnnnnssnnnnnnnnsnnnsnnnnnsnnnnHn1 GTTGAAGCGCGACCGTAAGCAAACTGCATACCGACGCCGGGGCGCCGCATCACTAACTACCATTGGACGTGCTGCAGGCAAGGCCGGHn2 ........C.....A........G....................................T....C....A..............C.Hb1 .......................G..T...........A..................G............A....G.........C.Hb2 ..................................................T.........T.........A................Hr1 .......................G.........................C..........T.........A..............C.Lc1 .......................G........................G.......T...T.C..C....A..............C.Lc3 ..............A........G........................G.......T...T.C..C....A..............C.Lc2 .....T..........................................G.....G.T........C....A................Ma1 ....................................T...........G.....G.T.............A..............C.Au1 .......................G........................G.....................A................Au2 ........CT....A........G........................G......G.........C....A..............C.Ps1 A....T.................G........................G.....G.T........C....A................Ps2 .....T.................G........................G.....G.T........C....A................Dc1 ....G.......A..........A........A......TCC......G.G.....T..TT.........A.............T..Dc2 ....G.......A..........A........A...............G.......T..TT.........A.............T..Dc3 ............A..........A........................G.....G.T...T.........A..............C.Dc4 ............A...................................G.....G.T.............A..............C.Dc5 .......................A........................G.G.....T...T.........A................Dc6 ........................C.............................G.T...T.........A..............C.Pl1 ....................................T.................G.T.............A..............C.Pl2 ................................................G.....G.T.............A..............C.Td1 .....................................T................................A..............C.Cl1 ............A................G..............................T.........AA.............C.Cl2 ............A....................................G....G.T...G.........AA.............C.Se1 ............A.........TG....................................T........................C.Se2 ............A.........TG....................................T......................G...Xo1 ............................................................T.........A..............C.Cf1 ............................C...............................T.........AA.............C.Ac1 .A....CAAG.C....G......G.A.......C.AT......A...AC..G.CG.TA..TG..CC..TAAAAA..AAAT.CCT..ALo1 ..C.C.CAAGAC.....T.....G.........C.AT......A...AC..GGCG.TA..TG..CC.GTAAAAAA.AAAT.CCT..ANb1 ....C..AAG.C.....T.....G.........CGA.........A...A....G.T...T....C..TAAAAA..A....C.T.C.Nl1 ....C...AG.C....GT..G..G.......A...AT........AT.........T...T...CCG.TAAAAA..A.A..C.T...Vm1 ....C..AAG.C.....T.....G.......A...A.........A........G.T...T....C..TAAAAA..A....C.T.C.Sc1 .....................G....................................G.T..C......AA...............Tb1 ...C.........AAA...A.......G.....C.A......G.T..A............T..C......AA......A......C.On1 ..................A.........................T..A.......G..G.T.........AA........G..T.C.On2 .............AAA...A.......G..A..C.A......G.T..A............T..C......AA......A......C.

Fig. 2 A nucleotide alignment of the cichlid c-type lysozyme.

Nucleotide sites are numbered according to the sequence of the

cichlid c-type lysozyme. The dots and letters indicate conserved

and variable nucleotides, respectively, compared with the

reference sequence on the top line. The abbreviations of each

species are as follows:O. niloticus (On), Steatocranus casuarius

Poll, 1939 (Sc), T. buttikoferi (Tb), Neolamprologus brichardi

Poll, 1974 (Nb), Neolamprologus leleupi Poll, 1956 (Nl),

Altolamprologus calvus Poll, 1978 (Ac), Variabilichromis

moorii Boulenger, 1898 (Vm), Lamprologus ocellatus

Steindachner, 1909 (Lo), Cyphotilapia frontosa Boulenger,

1906 (Cf), Xenotilapia ochrogenys Boulenger, 1914 (Xo), C.

leptosoma (Cl), S. erythrodon (Se), T. duboisi (Td), Aulonocara

sp. (Au), Maylandia lombardoi (synonym: Pseudotropheus

lombardoi) Burgess, 1977 (Pl), Pseudotropheus sp. (Ps), L.

caeruleus (Lc), D. compressiceps (Dc),Melanochromis auratus

Boulenger, 1897 (Ma), Haplochromis brownae Greenwood,

1962 (Hb), Haplochromis nigricans Boulenger, 1906 (Hn), and

H. sp. ‘redtail sheller’ (Hr). Nonsynonymous substitutions are

shown in a gray background


123

riverine species (On). Three Lake Tanganyika species

showed a single signal in each lane (Fig. 3B, T.

duboisi, Se, and A. calvus). Each of one Lakes Malawi

and Victoria species showed four and two signals,

respectively (Fig. 3B). In total, one riverine, four Lake

Tanganyika, two Lake Malawi, and one Lake Victoria

species possess two, one, four, and two copies of the

lysozyme gene, respectively. Based on the phyloge-

netic relationships among African cichlids (Salzburger

et al., 2002, 2005; Terai et al., 2003) and the Southern

blot results, we inferred the evolutionary transitions in

lysozyme copy number (Fig. 4).

We detected multiple haplotypes of lysozyme

genes from a Lake Malawi species. Using the primers

in the conserved regions among haplotypes from six

Lake Malawi species, we amplified DNA fragments

from cDNA (from exons 1 to 3) and genomic DNA

(from exons 2 to 3 with intron 2) (Fig. 1). These DNA

fragments were amplified using cDNA from D.

compressiceps#2, and genomic DNA from D. com-

pressiceps#6 (Table 1). We cloned and determined 28

and 24 sequences from cDNA and genomic DNA

templates, respectively, and found each of five hap-

lotypes from cDNA and genomic DNA (Fig. 5;

Table 1).

The evolution of lysozyme in cichlids

We identified the location of each variable amino acid

position using the 3D structure of hen egg white c-type

lysozyme. As shown in Fig. 6A, 45 out of 47 variable

amino acid positions were located on the surface of

lysozyme (shown in light-gray, nonsurface replace-

ments are surrounded by a rectangle in Fig. 6B).

D. com

pres

sicep

s

H.

T. du

boisi

S. e

ryth

rodo

n

A. ca

lvus

O. nilo

ticus

L. caeruleus (L. Malawi)

C. leptosoma (L. Tanganyika)

E: EcoRI

H: HindIII

P: PstI

E H P E H P

(L. Victoria) (L. Malawi) (L. Tanganyika) (L. Tanganyika) (L. Tanganyika) (Riverine)

(A)

(B)

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

2 kb

9.4 kb

PE PE PE PE PE PE

Fig. 3 The results of Southern blot analysis. Genomic DNAswere digested by EcoRI,HindIII, andPstI (A), or EcoRI and PstI (B). Thepositions of the DNA marker are shown by arrows


123

Among 45 surface replacement positions, 35 positions

substantially changed the charge of residues (posi-

tively charged–uncharged, positively charged–nega-

tively charged, negatively charged–uncharged, or vice

versa) (Fig. 6B, highlighted in gray). Among

sequences from Lakes Malawi and Victoria species,

12 replacements (marked by asterisks in Fig. 6B) out

of 19 surface replacements had a large effect on the

charge of residues.

We constructed a phylogenetic tree using all sites

(Fig. 7). The branch lengths in the Lamprologini

lineage (highlighted in gray) appeared to be longer

than the other branches in the phylogenetic tree

(Fig. 7).

Discussion

Isolation and characterization of cichlid lysozyme

In fish, the innate immune system is important for

defense against bacteria in various water environ-

ments (Saurabh & Sahoo, 2008). Lysozyme is

involved in the innate immune system; therefore, we

investigated the evolution of the lysozyme gene during

the radiation of East African cichlids. We determined

37 different sequences from Great Lakes and riverine

species. Among 87 variable sites, 64 were nonsyn-

onymous sites (Fig. 2), suggesting that this gene has

evolved with amino acid replacements during radia-

tion. Surprisingly, we isolated more than two lyso-

zyme haplotypes within a species of Lake Malawi

cichlids (L. caeruleus and Dc), whereas one or two

haplotypes were detected in each Lake Tanganyika

cichlid (Fig. 2; Table 1). This result raised the possi-

bility that the lysozyme copy number may differ

among cichlid species; therefore, we further analyzed

the gene copy number.

Lysozyme copy number varied during evolution

of cichlids

Based on the results of Southern blot analysis, the copy

numbers of lysozyme gene were estimated as two, one,

four, and two in one riverine, four Lake Tanganyika,

two Lake Malawi, and one Lake Victoria species,

respectively. This estimation includes a possibility of

underestimation of copy numbers, because Southern

blot analysis would not distinguish different copies in

the restriction enzyme fragments if they had the same

size. The sequence analysis from a single individual of

Lake Malawi species also supported the multicopy of

lysozyme genes. Each of the five haplotypes was

determined from cDNA and genomic DNA of Dc

M. auratusD. compressiceps

L. caeruleus

Pseudotropheus. sp.P. lombardoiAulonocara sp.

H. brownae

H. nigricansH.

T. duboisi

S. erythrodonC. frontosa

N. brichardiN. leleupi

L. ocellatus

X. ochrogenys

A. calvus

C. leptosoma

V. moorii

T. buttikoferiS. casuariusO. niloticus

gene duplication

gene duplication(s)

Lake Victoria

Lake Malawi

Lake Tanganyika

RiverineLam

prologini

Copy numbers

4

4

2

1

2

1*

1

1

Fig. 4 The positions of gene duplications on the phylogenetic

tree of cichlids. The lysozyme gene copy numbers based on

Southern blot and sequence analyses are shown on the left side

of each species. The positions of gene duplications are shown on

the branches of the phylogenetic tree. The phylogenetic tree is

based on previous studies (Salzburger et al., 2002, 2005; Terai

et al., 2003). *The possibility of two copies in C. leptosoma was

inferred from sequence analysis (see ‘‘Discussion’’ section)


123

(Fig. 5). The number of sequences from a single

individual indicates multiple copies of lysozyme

genes in the genome of Dc. Since cichlids are diploid

(Kuroiwa et al., 2013), we estimated that a minimum

of three lysozyme genes are present in the genome of

Dc, considering that all sequences are allelic varia-

tions. Two of five sequences amplified from both

cDNA and genomic DNA may be allelic variation,

because the results of Southern blot analysis showed

four copies of gene in Dc (Fig. 3B). Among the

nucleotide variation in six haplotypes determined

from five Dc individuals (Dc1–6), A at site 109

(109A), 111T, and 313C were not observed in

sequences determined from a single individual cDNA

(D. compressiceps#2), and 313C was not observed in

sequences from genomic DNA (D. compressiceps#6)

(Figs. 2, 5). The difference of nucleotide variation

between individuals may be due to the haplotype

variation within a species.

The copy number of the lysozyme gene in the

whole-genome sequence data from a Lake Malawi

cichlid did not match our Southern blot results from

Lake Malawi species (Fig. 3). The genome sequence

of Maylandia zebra Boulenger, 1899 (M_ze-

bra_UMD1: http://www.ncbi.nlm.nih.gov/) contains

two copies of the lysozyme gene (XP_014270133,

XP_014263224).Wemapped the short read sequences

(SRR077291) that were used for the assembly of the

M. zebra genome on the genome sequence (aligned

throughout the genome). In the two lysozyme genes,

the variants among Dc individuals at position 109,

257, 286, 299, 313, and 416 (Fig. 5) were observed at

the corresponding sites in the aligned short read

sequences. This result suggests a possibility that some

lysozyme genes are missing from the genome assem-

bly, because highly similar multicopy genes are dif-

ficult to assemble (Mariano et al., 2015).

To amplify the full-length of lysozyme coding

region from cDNA, we used a set of primers (LysF1

and LysR1) locating on the 50- and 30 UTRs for all

species used in this study. In this case, there was a

possibility that we could not amplify particular

haplotypes with mutations in primer biding positions,

indicating the possibility of underestimation of the

number of haplotypes.

In Lake Tanganyika species, two haplotypes were

determined from Cl (Cl1 and Cl2) and Se (Se1 and

Se2) (Fig. 2; Table 1), while a single band was

observed from each species (Fig. 3A, B). In the case

of Se, two haplotypes (Se1 and Se2) may represent

allelic variants, because the two haplotypes clustered

together in the phylogenetic tree (Fig. 7), and the

difference between these two haplotypes was only

one mutation (Fig. 2). In contrast, two haplotypes

from Cl (Cl1 and Cl2) were distantly related in the

tree (Fig. 7), and the difference between two

haplotypes was five mutations (Fig. 2). According

to these results, Cl seemed to possess two copies of

lysozyme gene. Lysozyme is one of the immune

system genes, and synonymous substitutions (posi-

tions 69 and 288) as well as nonsynonymous

substitutions in the sequences from Cl were shared

among species from different lineages (Fig. 2).

Therefore, we could not rule out the possibility that

the distantly related haplotypes of lysozyme have

been retained from an ancestral lineage, similar to

what has been previously reported for class II major

111222222 2344 46018334578 9112 99912694736 9364Dc1 GAATATCCGGA----------TTTGDc2 .....GGG.C.----------....Dc3 A...GGGG.CG----------C.CCDc4 A.C.GGGG.CG----------CCCCDc5 AC..GGGG...----------C.C.Dc6 ACCCGGGGACG----------C.CC

c1 ..CC.......----------..--c6 ..CC......G----------..--c7 ACCCGGGGACG----------C.--c12 ACCC......G----------..--c25 ACCC.......----------..--

g3 ----GGGGACG-intron 2-C.--g5 ----.GGG.C.-intron 2-..--

g15 ----....ACG-intron 2-C.--g17 ----GGGG.CG-intron 2-C.--

Nucleotide site

cDN

AG

enom

ic D

NA

Fig. 5 An alignment of all variable amino acid positions of

lysozyme haplotypes from D. compressiceps. The nucleotide

sites are shown on the top of the alignment. Dots indicate the

nucleotides that are identical with those in the top line.

HaplotypesDc1–6 were determined from fiveD. compressiceps

individuals. Haplotypes c1–25 were determined from cDNA of

a single individual (D. compressiceps#2). Haplotypes g3–17

were determined from genomic DNA of a single individual (D.

compressiceps#6)


123

http://www.ncbi.nlm.nih.gov/

histocompatibility complex genes in Lake Malawi

cichlids (Klein et al., 1993).

According to the results of Southern blot and

sequence analyses, the copy number of lysozyme

varied between one and four in African cichlids. The

evolutionary transitions in lysozyme copy number was

inferred based on the phylogenetic relationships

among African cichlids (Salzburger et al.,

2002, 2005; Terai et al., 2003) and the Southern blot

(Fig. 3) and sequence analyses. After colonization in

Lake Tanganyika, some species reinvaded riverine

habitats and their progeny independently colonized

Lakes Malawi and Victoria (Salzburger et al., 2005).

The gene duplications may have occurred in the

genomes of reinvaders and the ancestral species of

Lake Malawi (Fig. 4).

The evolution of lysozyme during cichlid adaptive

radiation

The adaptive evolution of lysozyme for optimum

enzymatic activity in various pH environments has

been reported (Stewart et al., 1987; Jolles et al., 1990;

Swanson et al., 1991; Irwin et al., 1992; Irwin, 1995;

Kornegay, 1996). Cichlid species are distributed in

lakes and rivers with pH values ranging from 6.0 to

10.0 (Fryer & Iles, 1972) in Africa. Therefore, we

assumed that the lysozyme gene might have evolved

rapidly to gain enzymatic activity in tissues facing

water environments with different pH values and salt

concentrations during radiation in different rivers and

lakes in East Africa.

Lake Victoria

Lake Malawi

Lake Tanganyika

Riverine

Lam

prol

ogin

i

amino acid position

(A)

(B) 1111111111 1111111 122222333 3345556667 7888889999 0001122223 3333334 7701367016 7897891347 9023461267 0585603780 1235692Hn1 FKERDRKANY RGKKATRTDR WCDRRHCNNV TPNRETVARQ GQDRPSGHn2 ...P.H.... G......... .......... .S..Q..T.. ......RHb1 .......... G........H .........G .......T.R ......RHb2 .......... .......... .......... .S.....T.. .......Hr1 .......... G......... .......... .S.....T.. ......RLc1 .......... G......... .....R.... .S..Q..T.. ......RLc3 .....H.... G......... .....R.... .S..Q..T.. ......RLc2 .I........ .......... .....R..D. ....Q..T.. .......Ma1 .......... .......... .....R..D. .......T.. ......RAu1 .......... G......... .....R.... .......T.. .......Au2 ...P.H.... G......... .....R..S. ....Q..T.. ......RPs1 .I........ G......... .....R..D. ....Q..T.. .......Ps2 .I........ G......... .....R..D. ....Q..T.. .......Dc1 .E..E..... S.....H... LSH..RW... IS.....T.. .....F.Dc2 .E..E..... S.....H... .....R.... IS.....T.. .....F.Dc3 ....E..... S......... .....R..D. .S.....T.. ......RDc4 ....E..... .......... .....R..D. .......T.. ......RDc5 .......... S......... .....RW... .S.....T.. .......Dc6 .......... .......... ........D. .S.....T.. ......RPl1 .......... .......... ........D. .......T.. ......RPl2 .......... .......... .....R..D. .......T.. ......RTd1 .......... .......... .......... .......T.. ......RCl1 ....E..... ...E...... .......... .S.....N.. ......RCl2 ....E..... .......... .....Q..D. .A.....N.. ......RSe1 ....E....F G......... .......... .S........ ......RSe2 ....E....F G......... .......... .S........ ....A..Xo1 .......... .......... .......... .S.....T.. ......RCf1 .......... .......... .......... .S.....N.. ......RAc1 I.DKA.E... GD.....PN. ...H.T.DDE .SDTQ.YKH. DKVTS.DLo1 .QDKT.M... G......PN. ...H.T.EDE .SDTQRYKHK DKVTS.DNb1 .Q.KA.M... G......RN. ....SQ..D. .S..Q.YKH. ...TS.RNl1 .Q.QA.V... G....K..N. ....I..... .S.TQAYKH. .K.TS..Vm1 .Q.KA.M... G....K..N. ....S...D. .S..Q.YKH. ...TS.RSc1 ........S. .......... .......... AS.....N.. .......Tb1 .....K.E.. ..E....PN. ...G.N.... .S.....N.. .K....ROn1 .......... .......... .....N..S. AS.....N.. ...GS.ROn2 .....K.E.. ..E.E..PN. ...G.N.... .S.....N.. .K....R

* * * * * * * * * * * *

Fig. 6 Characteristics of

variable lysozyme amino

acids among cichlids. A The

variable amino acid

positions of lysozyme in the

3D structure. The variable

amino acids are shown in

light gray. Right and left

panels show the view from

opposite sides. B An amino

acid alignment of the cichlid

c-type lysozyme. The dots

and letters indicate

conserved and variable

amino acids, respectively,

compared with the reference

on the top line. The

abbreviations for each

species are the same as in

Fig. 2. Amino acid

substitutions that change the

charge of residues are shown

in a gray background.

Nonsurface replacements

are surrounded by a

rectangle. The replacements

that occurred in Lakes

Malawi and Victoria

lineages are marked by

asterisks


123

To test this possibility, we analyzed the positions of

variable amino acids with respect to the structure of

lysozyme. If the amino acid substitutions affect the

function of lysozyme, the positions should localize on

the surface of the protein because there is a correlation

between the optimal pH for enzymatic activity and net

charge of surface amino acids (Pooart et al., 2005) that

is changed by surface amino acid residue composition.

Therefore, we identified the location of each variable

amino acid position using the 3D structure of hen egg

white c-type lysozyme. As shown in Fig. 6A, 45 out of

47 variable amino acid positions were located on the

surface of lysozyme, and 35 positions substantially

changed the charge of residues (Fig. 6B, highlighted

in gray). These results suggest that the replacements

localized on the surface may not affect the structure of

lysozyme but may change the maximal enzymatic

activity based on the net charge of surface amino acids

optimizing for different pH values. The gene duplica-

tions may have occurred in the genomes of the

ancestral species of Lakes Malawi and Victoria

(Fig. 4), and 12 replacements (marked by asterisks

in Fig. 6B) out of 19 surface replacements had a large

effect on the charge of residues among sequences from

Lakes Malawi and Victoria species. This result

suggests that the net charge of surface amino acids

changed after gene duplications occurred in these

lineages.

What was the role of the lysozyme substitutions in

the cichlid radiation? Given the function of lysozyme

in the innate immune system, the substitutions may

have affected defense against bacteria in various

habitats. The branch lengths in the Lamprologini

lineage (highlighted in gray) appeared to be longer

Lamprologini (L. Tanganyika)

Lakes Tanganyika

Malawi Victoria

Riverine

Xo1 Se2

Se1 Hn1

Hb2 Hb1

Au1 Td1 Hr1

Dc4 Pl2

Ma1 Pl1

Lc2 Ps1

Ps2 Dc3 Dc5

Dc1 Dc2

Cl2 Dc6

Au2 Hn2 Lc1

Lc3 Nb1

Vm1 Nl1

Ac1 Lo1

Cf1 Cl1 Sc1

On1 On2 Tb1

99

100

100

96

0.01

: Oreochromis niloticus: Oreochromis niloticus

: Steatocranus casuarius

: Tilapia buttikoferi

: Neolamprologus brichardi

: Altolamprologus calvus

: Variabilichromis moorii

: Lamprologus ocellatus: Cyphotilapia frontosa

: Cyprichromis leptosoma

: Cyprichromis leptosoma

: Spathodus erythrodon: Spathodus erythrodon

: Tropheus duboisi

: Aulonocara sp.

: Aulonocara sp.

: Pseudotropheus lombardoi

: Pseudotropheus lombardoi

: Labidochromis caeruleus: Labidochromis caeruleus

: Labidochromis caeruleus: Pseudotropheus sp.

: Pseudotropheus sp.

: Dimidiochromis compressiceps

: Dimidiochromis compressiceps: Dimidiochromis compressiceps

: Dimidiochromis compressiceps: Dimidiochromis compressiceps

: Dimidiochromis compressiceps

: Melanochromis auratus

: Haplochromis brownae: Haplochromis brownae

: Haplochromis nigricans

: Haplochromis nigricans

: Haplochromis

: Xenotilapia ochrogenys

: Neolamprologus leleupi

Fig. 7 A phylogenetic tree of lysozyme haplotypes. Phyloge-

netic inference employed neighbor joining with 1,000 bootstrap

replicates using all sites of the full-length region of lysozyme

cDNA (429 bp). Scale bar represents 0.01 substitutions per site.

The substrate breeding Lamprologini are highlighted in gray


123

than the other branches (Fig. 7). The amino acid

replacements in this lineage may be related to

certain living environments or traits specific to

Lamprologini species. All the species in this tribe

spawn eggs on the surface of the rock or inside the

empty shells of Neothauma tanganyikense Smith,

1880 (substrate-brooder) (Coulter, 1991; Salzburger

et al., 2002; Clabaut et al,. 2005; Koblmuller et al.,

2008a; Sturmbauer et al., 2010), where the risk of

bacterial infections may be high. However, the

correlation between egg spawning and the evolution

of lysozyme is unlikely, because all the haplotypes

were isolated from adult cDNAs. The other envi-

ronments such as water condition, depth, and

habitat may not be largely different between

Lamprologini and other Lake Tanganyika species.

The functional analysis of lysozyme from haplo-

types with long branches will be a key to find the

role of this gene in Lamprologini species.

In this study, we demonstrated a possible role of

gene duplications and amino acid replacements of

lysozyme that may contribute to the defense mecha-

nism against bacteria in environments with different

salt concentrations, pH values, temperatures, and

organic substances. Although the knowledge of the

role for gene duplication is still limited, the cichlid

genome sequences will facilitate the identification of

candidate genes with copy number variation among

lineages of African cichlids. The functional analysis of

the duplicated gene products will demonstrate the

importance of gene duplication during the adaptive

radiation of cichlid fish.

Acknowledgments This work was supported by the Ministry

of Education, Culture, Sports, Science, and Technology of Japan

Grants to Y.T. (Nos. 23570269 and 26440209), an internal

SOKENDAI Grant to Y.T., and the Center for the Promotion of

Integrated Sciences (CPIS) of SOKENDAI Grant to Y.T. I thank

Dr. Tetsumi Takahashi (Graduate School of Science, Kyoto

University, Japan; present address: Institute of Natural and

Environmental Sciences, University of Hyogo) for identification

of Lake Tanganyika cichlids, and Dr. Norihiro Okada (Graduate

School of Bioscience and Biotechnology, Tokyo Institute of

Technology; present address: Foundation of Advancement of

International Science, Japan; National Cheng Kung University,

Taiwan) for providing laboratory space and experimental

equipment.

Author contributions ST.K.: determination and analysis of

lysozyme sequences, manuscript editing. H.T.: manuscript

editing. Y.T.: research concept, research planning, all experi-

ments, data analysis, and manuscript preparation.

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