Phylogeography of the Maleo Senkawor ( Macrocephalon maleo Sal. Muller 1846) Based on Cytochrome B Gene in Sulawesi And Their Sex Determination using Molecular Sexing

ABSTRACT


INTRODUCTION
The Maleo Senkawor bird (Macrocephalon maleo) is a species of the Megapodiidae family endemic in Sulawesi island of Indonesia and threatened species by extinction.Many populations were lost due to overexploitation of their eggs and also lost connectivity between the forest and nesting sites (Arista et al. 2015;Froese & Mustari 2019).
The dispersal of M. maleo in Sulawesi formed population groups in nature.Thus, it is suspected that there has been local adaptation and contributed to the emergence of population diversity in each geographical distribution area.There is a need application of genetic marker candidate that able to reveal variations in the interpopulation and conserve requirements for nucleotides characterizing the species.The Cytochromeb (Cyt-b) gene is one of the genetic markers that have potential to be applied in this study.The Cytochrome-b gene is widely used as a marker of differences in geographic distribution and population origin of a species (Çoraman et al. 2013;Ibis et al. 2014;Petrova et al. 2014;Yong et al. 2015;Lapinski et al. 2016;Kato et al. 2016;Anucherngchai et al. 2019;Amiri et al. 2021).
Sources of DNA in birds that are commonly used for genetic analysis can be obtained through non-invasive such as feathers, eggshells, and feces (Begović et al. 2017;Kaunisto et al. 2017;Maia et al. 2017;Drahulian et al. 2018;Rytkönen et al. 2019;Peters et al. 2020;Demarchi et al. 2022).In the present study, DNA was obtained from the feather and eggshell material of the M. maleo bird.The use of non-invasive samples was more safe for M. maleo compared using a blood sample.
Differentiation and identification of species as well as sex ratios are important steps in conservative interventions (Awad et al. 2015).Therefore, research involving molecular in sex determination of M. maleo is important to determine the range of sex ratios in both natural and captive populations.Accurate sex determination is crucial for population development so that it can support the success of conservation programs for endangered species (Purwaningrum et al. 2019).Besides, the activity of egg taking and forest encroachment carried out by humans had a significant effect (87.94%) on the decline in the population of this species (Arista et al. 2015) and also large egg size (length 10.18 cm; width 6.12 cm; weight 211.70 grams) (Widnyanya 2017) attractive for the community to be used as a food.
The Maleo Senkawor is one of the species that have similar phenotypic characteristics between sex.Some basic methods that can be used for sex identification include differences in morphometric characteristics, laparoscopy for sex determination, sex chromosome examination, behavioral observations, and the presence of brooding patches.However, these methods have disadvantages.Sex identification through molecular techniques is more accurate than the other methods.The use of Polymerase Chain Reaction (PCR) method by utilizing specific primers for sex determination has been commonly used in various bird species (Khaerunnisa et al. 2013;Çakmak et al. 2017;Purwaningrum et al. 2019;Osman et al. 2020), but not much has been done on M. maleo, especially using materials from feathers and egg shells.One of the genes used in sex determination is Chromo Helicase DNA-Binding (CHD).This CHD gene was amplified by primer 2550F-2781R (Fridolfsson and Ellegren 1999).
The aimed of this study was therefore to reveal the M. maleo kinship relationships in different geographic areas in Sulawesi through the use of the gene marker Cytochrome-b (Cyt-b) from Mitochondria DNA with fragment size (903 bp).This Study was also aimed to determine the sex ratio and to examine the reliability of primers in amplification CHD gene with DNA material from feather and eggshell samples of M. maleo.The results are expected to support the conservation and development of the M. maleo population in Sulawesi island of Indonesia.

Study area
This research was carried out from July 2019 to January 2021.A total of 15 samples were collected, consisting of 14 feather samples and 1 eggshell sample (hatched).The research location covered 3 regions of North Sulawesi (7 samples), Southeast Sulawesi (5 samples), and Central Sulawesi (3 samples).

Total DNA isolation
Total DNA isolation was carried out from feathers and eggshells using the Dneasy® Blood and Tissue Kit (Cat. No. 69504).The procedure used followed on the Spin-Column Protocol procedure from Qiagen (2003) which has been modified by addition of buffer A and buffer B. The sample was then incubated at a temperature of 56 °C (overnight).

Amplification and aequencing of PCR products
The DNA replication process of the Cyt-b gene and CHD gene was carried out through the Polymerase Chain Reaction (PCR) technique.A pair of Cyt-b gene primers were designed based on the M. maleo genome data in GenBank (access code: MW574376.1).The primers were designed through the primer3 program (http://bio-info.ut.ee/primer3-0.4.0/primer3).Meanwhile, the primers used to amplify the CHD gene are universal primers, namely 2550F/2718R designed by (Fridolfsson and Ellegren 1999).The primers can be seen in the following Table 1.
PCR conditions during the Cyt-b gene amplification process included Pre-denaturation at 94°C for 3 minutes, Denaturation at 94°C for 45 seconds, annealing at 54,45°C for 45 seconds, extension at 72°C for 1 minute with a total of 35 cycles, and post extension 72°C for 6 minutes.Meanwhile, the PCR conditions during the CHD gene amplification process included predenaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 51,5°C for 45 seconds, an extension at 72°C for 1.5 minutes with a total of 35 cycles, and post extension at 72°C for 10 minutes.Detection of PCR products between the Cyt-b gene and the CHD gene was carried out through electrophoresis on 1.2% agarose gel using TBE-1x buffer.Good DNA bands on target were sequenced through commercial services (First Base Malaysia).Identification of the sex of each individual was based on the number of emerged DNA bands.One (1) visible band was identified as a male individual and two (2) bands were identified as female individuals (Vucicevic et al. 2013;Çakmak et al. 2017).

Data analysis
Sequence data of the Cyt-b gene and the CHD gene were aligned using MEGA (Molecular Evolutionary Genetics Analysis) software version 7.0 (Kumar et al. 2016).Furthermore, the primer was used to correct the nucleotide sequence to obtain a good sequence.Subsequent sequences were BLAST-n at the National Center for Biotechnology Information (NCBI) site.Reconstruction of the phylogenetic tree was carried out using the Neighbor-Joining (NJ) and UPGMA Unweighted Pair Group Method with Arithmetic Average (UPGMA) Kimura 2parameter model and pdistance at 1000 bootstraps repetitions.Observation of haplotype diversity used the DNAsp program version 6 (Rozas et al. 2017) and construction of phylogenetic networks was carried out using the Median-Joining method (Bandelt et al. 1999) using the Network program version 10.2 (Fluxus Technology Ltd c2021).Furthermore, 2 sequences from GenBank as an ingroup, namely M. maleo (accession number: AM236881.1 and MW574376.1),and 1 sequence from GenBank as an outgroup, namely Alectura lathami (accession number: AY346091.1).

DNA purity
Quantification of the extracted genomic DNA using the Dneasy® Blood and Tissue Kit (Cat.No. 69504) was carried out to obtain the concentration and purity values of DNA before PCR was performed.The average value of the concentration and purity of DNA from samples of feathers and eggshells of M. maleo can be seen in Table 2.
The quantification results in Table 2 showed that the average level of DNA purity is good (1.8-1.9).DNA can be said to be good if the value of the A260/A280 ratio is between 1.8-2.0(Işçi et al. 2014;Yalçınkaya et al. 2017).

BLASTn result analysis
Sequence data were obtained after being BLASTn on the NCBI site to determine the similarity value to the The search results can be seen in (Tables 3 and Table 4).Table 3 showed that the samples analyzed are Maleo Senkawor birds (M.maleo) with a closeness of more than 99%.
Data in Table 4 and Figure 1 showed that seven (7) individuals in the population from North Sulawesi were identified as male.The population from Southeast Sulawesi identified 1 female and four (4) males.Meanwhile, in the population of Central Sulawesi, one (1) female and two (2) male individuals were identified.These results indicated that the ratio of male and female individuals in the analyzed sample is 6,5:1.
Sex determination of M. maleo showed that the sequence sizes of the CHDZ and CHDW genes were 586 bp and 436 bp, respectively (Figure 1).In Figure 1, it can be seen that the female individual with 2 bands (CHDZ/CHDW) in the samples from Southeast Sulawesi (MKT1) and Central Sulawesi (MS3) had 586 bp (CHDZ) and 436 bp (CHDW), while the male individual (CHDZ/CHDZ) has only a single band (586 bp).

Haplotype
Based on the data in Tables 5 and Table 4 haplotypes consisted of 3 interpopulation haplotypes and 1 haplotype based on sequences from GenBank.

Amino acid sequence polymorphism
The translation results of 903 bp of nucleotide sequences of the partial Cyt-b gene of M. maleo obtained 301 amino acid (AA) sites.The results of this analysis provided information that there are 298 conserved AA sites and 2 varied AA encoding sites.The various AA sites are at sites 297 and 300 which are non-synonymous AA (coding nucleotides changed, amino acids changed).Changes occured at 2 sites (297 and 300) are transitional substitutions that occured in pyrimidine bases (Thymine to Cytosine or vice versa) and also in purine bases (Guanine to Adenine or vice versa).Substitution transition occurs at the triplet of codon 2 (890th site of T>C) and the triplet of codon 1 (898th site of G>A).AA substitution occurs at site 297 (from L>P or Leucine to Proline) AA substitution at this site occurs only in populations from North Sulawesi.Meanwhile, the 300th site (A>T or Alanine to Threonine) occurred in populations from North Sulawesi and Central Sulawesi (Table 6).

Central Sulawesi . T
The dot symbol ( .) is an identical sequence to M. maleo from Genbank (AM236881.

Phylogenetic tree construction
The phylogenetic tree topology of M. maleo interpopulation in Sulawesi and 2 reference sequences from GenBank (AM236881.1 and MW574376.1)based on nucleotide sequences of the Cyt-b gene are still in the same cluster (1 cluster).Even though they are in the same cluster, it appears that the populations of M. maleo from North Sulawesi and Central Sulawesi tend to have a closer kinship relationship (Figure 3).The phylogenetic tree topology based on AA also shows the same pattern as the nucleotide sequences of the Cyt-b gene and it is known that populations from North Sulawesi and Central Sulawesi tend to have a closer kinship than populations from Southeast Sulawesi (Figure 4)

Discussion
Variations in nucleotide bases in the intrapopulation of M. maleo indicated that there is no substitution.Meanwhile, the interpopulation occurs based on a transitional substitution.The results of the analysis showed that transition substitution occured 4 times and no transversion substitution occurs.In Luscinia calliope, the dominant transition substitution is based on the Cytb gene (Spiridonova et al. 2013).The occurrence of nucleotide base substitution in the interpopulation resulted in the percentage of M. maleo genetic distance between North, Southeast, and Central Sulawesi (0.2% -0.3%.).The low value of the genetic distance of M. maleo in the interpopulation indicates that there are no significant genetic differences between the populations of North, Southeast, and Central Sulawesi.This result is presumably because genetic mixing between subpopulations (gene flow) continues to occur so that the differences between different geographic distributions are not significant (the geographic distribution barrier is relatively small).However, geographical variations have occurred between the origin of the geographic distribution of North Sulawesi, Central Sulawesi, and Southeast Sulawesi.
The value of nucleotide diversity and haplotype diversity based on intrapopulation obtained the same value, namely 0.00000 (0%), the low value is suspected whether the population size in each habitat is low or indeed the genetic diversity is low in nature.However, these results provided information that there is a negative impact that could threaten the survival of the M. maleo population due to the low intrapopulation nucleotide and haplotype diversity.Low genetic diversity may appear in small populations through inbreeding, thereby triggering negative traits, including decreased fitness, adaptation to environmental changes, reproductive problems, disease susceptibility, and other similar conditions that can reduce population persistence (Pekkala et al. 2014;Fünfstück and Vigilant 2015;Hedrick and Garcia-Dorado 2016;Harrisson et al. 2019).
Based on the pattern formed in the haplotype network, the Southeast Sulawesi sub-population and the sequence from GenBank (MW574376.1)are combined into the same haplotype, namely Haplotype-2.This condition is because this sub-population is still composed of common nucleotides found in all populations.Meanwhile, the sub-populations of Central Sulawesi, North Sulawesi, and sequences from Genbank (AM236881.1)appeared to form separate subpopulations.
The existence of M. maleo in Sulawesi as a species in the Family Megapodiidae that has evolved, it might be thought to have originated from an ancestor in Australia.Megapodiidae existed in Australia in the late Oligocene and there is general agreement that Megapodiidae were isolated in Australia-Papua for a long time (Harris et al. 2014).In addition, M. maleo reaching the island of Sulawesi is inseparable from the process of forming this island.It is suspected that Sulawesi originated from Mainland Asia, Mainland Australia, and lands arising from the ocean floor during the Miocene to Pleistocene epochs (Hall 2001;Shekelle and Leksono 2019).M. maleo colonization on Sulawesi Island is directly related to the geological formation and source of its initial population and evolution of development.
Based on the results of sex identification, it is known that from the 15 samples analyzed, 13 individuals were male and 2 individuals were female.The dominance of male individuals over female individuals occurred in 3 study population areas.The sex ratio plays an important role and influences sex roles and the reproductive system (Szekely et al. 2014).An unbalanced sex ratio in a small population can result in a decline of population that leads to the extinction of the species (Kus et al. 2017) and this certainly harms M. maleo as an endangered species.Sex determination using primers 2550F and 2718R (Fridolfsson and Ellegren 1999) was able to distinguish the sex of the M. maleo bird species with DNA sources from feathers and eggshells.The size of the CHDW and CHDZ fragments through the primary determination of 2550F/2718R varied, namely 440 bp -705 bp (Çakmak et al. 2017), 600 bp-100 bp (Ravindran et al. 2019).The results of this study were not significantly different from those that had been done previously on CHD-Z M. maleo (Yuda and Saputra 2021).
Habitat damage, especially in coastal areas in Sulawesi, which is one of the spawning habitats of M. maleo is very massive because it is used as an area for agriculture, aquaculture, and settlements.The decreasing number of nests of M. maleo is thought to be strongly influenced by the deterioration of conditions and the loss of the ideal nesting habitat for regeneration.Thus, the population declined and the limited geographical distribution of this animal are becoming increasingly limited and population development is increasingly worrying.Many areas where previously this animal species could be found, are now empty.In addition to habitat destruction, egg hunting is are also a serious threat to the survival of this species, because the egg size is large so it is interesting for human consumption.

CONCLUSION
Based on genetic markers, the Cyt-b gene is large (79% of total length) indicating that populations from North Sulawesi and Central Sulawesi have a closer kinship with a genetic distance marker of 0.2%.Meanwhile, the population from Southeast Sulawesi is seen to be separated from other populations with a genetic distance of 0.3%.Although they are still in the same cluster (populations of North Sulawesi and Central Sulawesi), each population has its haplotype, namely haplotype-3 in populations from North Sulawesi and haplotype-4 in populations from Central Sulawesi.The Southeast Sulawesi population belongs to the sequence from GenBank (MW574376.1)has its haplotype, namely haplotype-2, and is in a separated cluster.Primers 2550F and 2718R were able to amplify the CHD gene in M. Maleo with fragment sizes for CHDZ and CHD W, were 546 bp and 436 bp, respectively.Meanwhile, sex identification showed that the male sex was dominant over the female sex with a ratio of 6,5:1

Figure 1 .Figure 2 .
Figure 1.Differences in the size of the Chromo Helicase DNA-binding (CHD) gene in M. maleo.(M: DNA Ladder 100 bp); MT= Maleo Tambun (North Sulawesi); MKT= Maleo Kolaka Timur (Southeast Sulawesi); MBHM= Maleo Blok Hutan Mempaho (Southeast Sulawesi); MTB= Maleo Tanjung Batikolo (Southeast Sulawesi); MS= Maleo Saluki (Center Sulawesi) nucleotides are marked in black boxes; bold numbers represent nucleotide sequences based on the partial sequence of the Cyt-b gene; the number in brackets is the nucleotide sequence based on the complete genome sequence of the Cyt-b gene.

Figure 3 .
Figure 3.The relationship between populations of Macrocephalon maleo based on the Neighbor-Joining (NJ) method, bootstrap 1000 repetitions with the Kimura 2-parameter model on the nucleotide sequence of the Cyt-b gene

Figure 4 .
Figure 4.The population between relations of Macrocephalon maleo based on the UPGMA (Unwight Pair Group Method with Arithmetic Average) method, bootstrap 1000 repetition with the p-distance model in the amino acid (AA) sequence

Table 1 .
Primers used in the study

Table 2 .
Average concentration and purity of DNA

Table 3 .
Species identification based on BLASTn results in NCBI

Table 4 .
Sex determination based on BLASTn results in NCBI

Table 5 .
Nucleotide sequence polymorphisms (SNPs) of the M. maleo interpopulation based on the Cyt-b gene

Table 6 .
Codon coding sites and amino acids that changed in the Cyt-b gene of M. maleo