# Efficient Discrimination of Some Moss Species by Fourier Transform Infrared Spectroscopy and Chemometrics

Authors: Zhen Cao; Yongying Liu; Jiancheng Zhao
Journal: Journal of Spectroscopy
 (2014)

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## Abstract

Fourier transform infrared spectroscopy (FTIR) technique was used to classify 16 species from three moss families (Mielichhoferiaceae, Bryaceae, and Mniaceae). The FTIR spectra ranging from 4000 cm−1 to 400 cm−1 of the 16 species were obtained. To group the spectra according to their spectral similarity in a dendrogram, cluster analysis and principal component analysis (PCA) were performed. Cluster analysis combined with PCA was used to give a rough result of classification among the moss samples. However, some species belonging to the same genus exhibited very similar chemical components and similar FTIR spectra. Fourier self-deconvolution (FSD) was used to enhance the differences of the spectra. Discrete wavelet transform (DWT) was used to decompose the FTIR spectra of Mnium laevinerve and M. spinosum. Three scales were selected as the feature extracting space in the DWT domain. Results showed that FTIR spectroscopy combined with DWT was suitable for distinguishing different species of the same genus.


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## Body

## 1. Introduction

Mosses are perennial plants, typically 1 cm to 10 cm tall, usually occur in large tufts, and concentrate in groups. They possess a gametophyte-dominated life cycle. Spores are released from a sporophyte capsule at certain times. Mosses leaves commonly exhibit a spiral phyllotaxy. Rhizoids are often branched. Given that they do not have any root system and barely no cuticle, mosses obtain most of their nutrients from the atmosphere (wet and dry deposition) [1]. Because of their limited living forms, bryophytes usually grow in harsh habitats, where no vascular plants can live. With over 12,000 species recognized worldwide, mosses are one of the most speciose among plants [2]. They are distributed from the Arctic to the Antarctic and occupy a broad range of habitats. They show extensive morphological and anatomical diversification in both gametophyte and sporophyte organization [3].The taxonomic concepts and phylogenetic relations among genera of the families Mielichhoferiaceae, Bryaceae, and Mniaceae have given rise to much controversy in recent years [4]. Studies using nuclear, plastid, and mitochondrial sequences showed that many genera of the Mielichhoferioideae and Bryaceae families are polyphyletic [5–8]. Some species of Mielichhoferiaceae, Bryaceae, and Mniaceae are difficult to distinguish by traditional phytotaxonomic methods. For instance, the species of genusPohlia was originally placed in family Bryaceae according to the characteristics of the leaf cells [9]. A phylogenetic analysis of random amplified polymorphic DNA (RAPD) markers showed thatPohliawas more closely related to the genera of Mniaceae than Bryaceae [4]. However, Wang et al. declared thatPohlia should not be classified in the Mniaceae and was indicated to be a closer relative of generaMielichhofera (Mielichhoferioideae) [8, 10]. Fourier transform infrared (FTIR) spectroscopy is an alternative method for species discrimination, which has already been demonstrated for medicinal plants, bacteria, fungi, and microorganisms [11–14]. Although FTIR spectroscopy has made a significant contribution to the plant classification, reports on mosses taxonomy by this analytical approach are still rare [15].FTIR is an original spectroscopic technique used in investigating the functional groups of chemical components [16, 17]. It presents good sensitivity and a remarkable simplicity in sample preparation. Each FTIR spectrum of a compound can express a unique “fingerprint.” This feature enables FTIR spectroscopy to be used in classification of different samples or identification of unknown samples [18]. However, if differences in the chemical compositions are small, the FTIR spectra will be very similar. Then the analysis of using only FTIR spectra is insufficient to group a set of samples [19]. Chemometric methods combined with FTIR spectroscopy can compensate the defects in single FTIR spectral analysis [20]. The way of using chemometrics to make determination and classification of the confused species becomes an effective research object in the instrument analysis [14, 21]. In our study, the FTIR spectra of some species in families Mielichhoferiaceae, Bryaceae, and Mniaceae were similar. So we chose chemometric methods to further discriminate different species in the three families.Wavelet transform (WT) is a useful tool for a variety of signal processing applications. For example, WT is useful for the compression of digital image files, noise reduction, and pattern recognition. It provides a time-frequency representation of the signal. It was also developed to overcome the discrimination of nonstationary signals with different frequency features [22]. The signal to be analyzed is multiplied with a wavelet function; the analyzing ability for the same signal of wavelet coefficients is different at different scales. Therefore, the WT coefficients can be considered as the characteristics of the signal. A few features can reflect the major spectral information after a wavelet function; WT can be considered one of the most efficient chemometric methods [23].Cluster analysis and principal component analysis (PCA) are two other multivariate analyses that can be used to identify natural clustering pattern and group objects on the basis of similarities among the samples [24]. Cluster analysis and PCA are widely recognized as very powerful tools in obtaining information about relations within a dataset [25, 26].The present study aimed to evaluate the potential use of FTIR spectroscopy combined with cluster analysis and PCA technique for the discrimination of 16 moss species. Fourier self-deconvolution (FSD) and discrete WT (DWT) were used to investigate the variability of the chemical fingerprint among the species.


## 2. DWT

DWT is used to decompose a signal by using filters to extract interesting frequency resolution components within the signal. DWT possesses compact support in both time and frequency domains [27]. It is a signal-processing tool that is used in many engineering, scientific, and mathematical applications. DWT is used to analyze the signal at different frequency bands with different resolutions by decomposing the signal into a coarse approximation and detailed information [28]. DWT originated from the discretization of continuous WT (CWT), and the common discretization is dyadic. CWT is provided by
(2)
X
wt
(
τ
,
s
)
=
1
|
s
|
∫
-
∞
∞
x
(
t
)
Ψ
*
(
t
-
τ
s
)
d
t
,
where x
(
t
) is the signal to be analyzed and Ψ
*
(
t
) is the mother wavelet or the basis function. All of the wavelet functions used in the transformation are derived from the mother wavelet through translation and scaling.In CWT, the signals are analyzed using a set of basic functions, which are related to each other by simple scaling and translation. In the case of DWT, a time-scale representation of the digital signal is obtained using digital filtering techniques. The DWT is derived from the discretization of CWT(
τ
,
s
) and the most common discretization is dyadic. DWT is provided by
(2)
X
dwt
(
j
,
k
)
=
1
2
j
∫
-
∞
∞
x
(
t
)
Ψ
*
(
t
-
2
j
k
2
j
)
d
t
,
where τ and s are replaced by 2
j and 2
j
k. The DWT is computed by successive low-pass and high-pass filtering of the discrete time-domain signal. This computation is called the Mallat algorithm or Mallat-tree decomposition. Its significance is in the manner it connects the continuous-time multiresolution to discrete-time filters. With this approach, the time resolution becomes arbitrarily good at high frequencies, whereas the frequency resolution becomes arbitrarily good at low frequencies [29].


## 3. Materials and Methods

### 3.1. Sample Preparation

The 16 species of mosses were collected from Hebei, China, in August 2011. The environmental conditions and population sizes of the sample plants were similar. The geographic coordinates and altitudes are shown in Table1. The voucher specimens are deposited in the Herbarium of Hebei Normal University.Table 1
Description of the geographic coordinates and altitudes of the sample sources.
Species label number
Species
Genera
Family
Site
Altitude (m asl)
Geographic coordinates
Voucher specimen number
1
Pohlia  cruda
Pohlia
Mielichhoferiaceae
HY
1414
39°56′N114°57′E
2011253L
2
Pohlia  longicollis
HY
1294
39°56′N114°56′E
2011103L
3
Pohlia  minor
HY
1342
39°56′N114°57′E
2011305L
4
Pohlia  elongata
HY
1460
39°56′N114°58′E
2011016L
5
Bryum  argenteum
Bryum
Bryaceae
HY
1709
39°55′N114°57′E
2011154L
6
Bryum  uliginosum
HY
1713
39°55′N114°57′E
2011024L
7
Bryum  capillare
HY
1540
39°56′N114°58′E
20110620X
8
Mnium  spinosum
Mnium
Mniaceae
HY
1468
39°56′N114°59′E
20110151X
9
Mnium  laevinerve
HY
1507
39°56′N114°59′E
20110324X
10
Mnium  marginatum
HY
2625
39°54′N114°58′E
20110493X
11
Plagiomnium  acutum
Plagiomnium
HY
2483
39°54′N114°58′E
20110648X
12
Plagiomnium  succulentum
HY
1473
39°56′N114°58′E
20110480X
13
Plagiomnium  maximoviczii
HY
1469
39°55′N114°58′E
20110429X
14
Plagiomnium  arbusculum
HY
1517
39°55′N114°59′E
20110457X
15
Trachycystis  flagellaris
Trachycystis
HY
1583
39°56′N114°58′E
20110206X
16
Trachycystis  ussuriensis
HY
1585
39°56′N114°58′E
20110212X
HY: Yuxian county, Hebei province, China.To avoid problems caused by the influence of water on FTIR spectra, all of the samples were dried at 35°C in a drying oven for one week until a constant weight was achieved. The samples were ground into fine powder in an agate mortar and then sieved through 200 meshes (75μm).


### 3.2. Spectral Measurements

The FTIR spectra (range 4000 cm−1 to 400 cm−1, 4 cm−1 resolution, 32 scans) were obtained using a Bruker Optics (Ettlingen, Germany) Vertex70 FTIR spectrometer equipped with a DigiTect detector, which could prevent external signal disturbance and guarantee the highest signal-to-noise ratio. Conventional KBr pellet spectra were recorded from the sample. After grinding, 2 mg dried sample powder was mixed with 50 mg dried KBr power and then compressed with the use of an evacuable pellet die to produce a 13 mm diameter pellet. The pellets were compressed under the same mechanical pressure, and their FTIR spectra were obtained. The FTIR spectra of the 16 mosses samples were automatically baseline corrected. Background was recorded each time before the measurement and subtracted automatically by the software [30]. Each of the samples was measured three times and the averaged spectrum was obtained for further analysis.


### 3.3. Precision and Repeatability Test

To validate the method, the FTIR spectra of sample no. 11 (Plagiomnium acutum(Lindb.) T. Kop) were recorded. The precision test was determined by replicate records (5x) of the same sample. The repeatability test was analyzed by recording the same species from five different areas independently (Table 2). In statistics, the relative standard deviation (RSD or %RSD) is the absolute value of the coefficient of variation. It is often expressed as a percentage. The RSD is widely used in analytical chemistry to express the precision and repeatability of an assay. In this study, the mean value RSD of transmittances of the communal peaks was calculated.Table 2
Moss plant samples (Plagiomnium  acutum) collected from different places.
Sample number
Region
Altitude (m asl)
Time of collection
Voucher specimen number
P1
Pingshan, Hebei
1516
2011.05
C0215
P2
Pingshan, Hebei
1064
2011.05
C0287
P3
Yuxian, Hebei
981
2011.07
X0059
P4
Yuxian, Hebei
1821
2011.07
X0087
P5
Zhuolu, Hebei
1076
2011.08
X0125


### 3.4. Data Analysis

Spectral data were calculated with the use of OMINIC version 8 software (Thermo Fisher, Waltham, USA). Cluster analysis and PCA were conducted with the use of IBM SPSS 19.0 software. Cluster analysis is an undirected, unbiased statistical method used to analyze spectroscopic information and it has been used to sort the FTIR spectra into similar sets or groups [31]. Satisfactory results on FTIR spectra of complex biological molecules have been obtained with Ward’s algorithm, Euclidean distances, or correlation coefficient calculation as distance metrics [32, 33]. In the present study, 16 samples of mosses were selected for clustering. The 20 transmittance peaks in the range of 4000 cm−1 to 400 cm−1 were then used for PCA analysis [15]. In order to make the cluster analysis and PCA analysis more accurate, we chose 20 transmittance peaks in the range of 4000 cm−1 to 400 cm−1 for cluster analysis. The nearest neighbor and Pearson correlation were used to construct dendrograms. PCA is a multivariate statistical data reduction method. It transforms the original set of variables to a new set of uncorrelated variables called principal components (PCs) [34]. It also provides information on the major spectral components, in which dominant factors determine differences among samples [35–37]. PCA is used to extract the important features of the correlation matrix in terms of PCs. Only a few PCs are usually required to explain the majority of observed variance. PCA can be used as a chemometric method for FTIR analysis. The analysis can be presented as either two-dimensional (2D, two PCs) or three-dimensional (3D, three PCs) scatter plots [38]. The 20 selected transmittance peaks were used for PCA and the factor loading was plotted.FSD was performed using OMINIC version 8 software. DWT was performed using MATLAB 7.1 software. Daubechies wavelet possesses better exploration ability for signal singularity, which has functioned as analysis wavelet. One-dimensional stationary DWT can be performed to different samples [39].


## 3.1. Sample Preparation

The 16 species of mosses were collected from Hebei, China, in August 2011. The environmental conditions and population sizes of the sample plants were similar. The geographic coordinates and altitudes are shown in Table1. The voucher specimens are deposited in the Herbarium of Hebei Normal University.Table 1
Description of the geographic coordinates and altitudes of the sample sources.
Species label number
Species
Genera
Family
Site
Altitude (m asl)
Geographic coordinates
Voucher specimen number
1
Pohlia  cruda
Pohlia
Mielichhoferiaceae
HY
1414
39°56′N114°57′E
2011253L
2
Pohlia  longicollis
HY
1294
39°56′N114°56′E
2011103L
3
Pohlia  minor
HY
1342
39°56′N114°57′E
2011305L
4
Pohlia  elongata
HY
1460
39°56′N114°58′E
2011016L
5
Bryum  argenteum
Bryum
Bryaceae
HY
1709
39°55′N114°57′E
2011154L
6
Bryum  uliginosum
HY
1713
39°55′N114°57′E
2011024L
7
Bryum  capillare
HY
1540
39°56′N114°58′E
20110620X
8
Mnium  spinosum
Mnium
Mniaceae
HY
1468
39°56′N114°59′E
20110151X
9
Mnium  laevinerve
HY
1507
39°56′N114°59′E
20110324X
10
Mnium  marginatum
HY
2625
39°54′N114°58′E
20110493X
11
Plagiomnium  acutum
Plagiomnium
HY
2483
39°54′N114°58′E
20110648X
12
Plagiomnium  succulentum
HY
1473
39°56′N114°58′E
20110480X
13
Plagiomnium  maximoviczii
HY
1469
39°55′N114°58′E
20110429X
14
Plagiomnium  arbusculum
HY
1517
39°55′N114°59′E
20110457X
15
Trachycystis  flagellaris
Trachycystis
HY
1583
39°56′N114°58′E
20110206X
16
Trachycystis  ussuriensis
HY
1585
39°56′N114°58′E
20110212X
HY: Yuxian county, Hebei province, China.To avoid problems caused by the influence of water on FTIR spectra, all of the samples were dried at 35°C in a drying oven for one week until a constant weight was achieved. The samples were ground into fine powder in an agate mortar and then sieved through 200 meshes (75μm).


## 3.2. Spectral Measurements

The FTIR spectra (range 4000 cm−1 to 400 cm−1, 4 cm−1 resolution, 32 scans) were obtained using a Bruker Optics (Ettlingen, Germany) Vertex70 FTIR spectrometer equipped with a DigiTect detector, which could prevent external signal disturbance and guarantee the highest signal-to-noise ratio. Conventional KBr pellet spectra were recorded from the sample. After grinding, 2 mg dried sample powder was mixed with 50 mg dried KBr power and then compressed with the use of an evacuable pellet die to produce a 13 mm diameter pellet. The pellets were compressed under the same mechanical pressure, and their FTIR spectra were obtained. The FTIR spectra of the 16 mosses samples were automatically baseline corrected. Background was recorded each time before the measurement and subtracted automatically by the software [30]. Each of the samples was measured three times and the averaged spectrum was obtained for further analysis.


## 3.3. Precision and Repeatability Test

To validate the method, the FTIR spectra of sample no. 11 (Plagiomnium acutum(Lindb.) T. Kop) were recorded. The precision test was determined by replicate records (5x) of the same sample. The repeatability test was analyzed by recording the same species from five different areas independently (Table 2). In statistics, the relative standard deviation (RSD or %RSD) is the absolute value of the coefficient of variation. It is often expressed as a percentage. The RSD is widely used in analytical chemistry to express the precision and repeatability of an assay. In this study, the mean value RSD of transmittances of the communal peaks was calculated.Table 2
Moss plant samples (Plagiomnium  acutum) collected from different places.
Sample number
Region
Altitude (m asl)
Time of collection
Voucher specimen number
P1
Pingshan, Hebei
1516
2011.05
C0215
P2
Pingshan, Hebei
1064
2011.05
C0287
P3
Yuxian, Hebei
981
2011.07
X0059
P4
Yuxian, Hebei
1821
2011.07
X0087
P5
Zhuolu, Hebei
1076
2011.08
X0125


## 3.4. Data Analysis

Spectral data were calculated with the use of OMINIC version 8 software (Thermo Fisher, Waltham, USA). Cluster analysis and PCA were conducted with the use of IBM SPSS 19.0 software. Cluster analysis is an undirected, unbiased statistical method used to analyze spectroscopic information and it has been used to sort the FTIR spectra into similar sets or groups [31]. Satisfactory results on FTIR spectra of complex biological molecules have been obtained with Ward’s algorithm, Euclidean distances, or correlation coefficient calculation as distance metrics [32, 33]. In the present study, 16 samples of mosses were selected for clustering. The 20 transmittance peaks in the range of 4000 cm−1 to 400 cm−1 were then used for PCA analysis [15]. In order to make the cluster analysis and PCA analysis more accurate, we chose 20 transmittance peaks in the range of 4000 cm−1 to 400 cm−1 for cluster analysis. The nearest neighbor and Pearson correlation were used to construct dendrograms. PCA is a multivariate statistical data reduction method. It transforms the original set of variables to a new set of uncorrelated variables called principal components (PCs) [34]. It also provides information on the major spectral components, in which dominant factors determine differences among samples [35–37]. PCA is used to extract the important features of the correlation matrix in terms of PCs. Only a few PCs are usually required to explain the majority of observed variance. PCA can be used as a chemometric method for FTIR analysis. The analysis can be presented as either two-dimensional (2D, two PCs) or three-dimensional (3D, three PCs) scatter plots [38]. The 20 selected transmittance peaks were used for PCA and the factor loading was plotted.FSD was performed using OMINIC version 8 software. DWT was performed using MATLAB 7.1 software. Daubechies wavelet possesses better exploration ability for signal singularity, which has functioned as analysis wavelet. One-dimensional stationary DWT can be performed to different samples [39].


## 4. Results and Discussion

### 4.1. Validation of the Method

#### 4.1.1. Precision Test

The precision test was conducted by replicate measurements of the same sample (Plagiomnium acutum) for five times in a day. The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤0.51%.Table 3
FTIR transmittances of the characteristic common peaks of precision and repeatability tests onPlagiomnium  acutum.
Peak number
Transmittance % mean (RSD %)
PT
RT
1
13.386 (0.51)
13.635 (0.81)
2
28.041 (0.14)
29.163 (0.18)
3
52.856 (0.12)
53.932 (2.17)
4
13.969 (0.23)
14.306 (0.76)
5
22.608 (0.16)
21.652 (0.84)
6
10.738 (0.33)
10.560 (1.42)
7
29.045 (0.13)
30.145 (0.73)
8
27.033 (0.12)
27.145 (0.95)
9
25.063 (0.15)
25.852 (1.07)
10
23.617 (0.09)
24.444 (1.17)
Precision test (PT,n
=
5), repeatability test (RT, n
=
5).


#### 4.1.2. Repeatability Test

The repeatability of the method was assessed by analyzing five independently prepared samples ofPlagiomnium acutumfrom five different places using the same method (Table 2). The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤2.17%.The running results show that the FTIR transmittances of the characteristic common peaks of the same species are stable. The method is reliable and exhibits good repeatability; thus, it can be applied in the analysis of other moss samples.


### 4.2. FTIR Analysis

The FTIR spectra of the 16 samples of mosses were recorded in three sets: four Mielichhoferiaceae samples (Figure1(a)), three Bryaceae samples (Figure 1(b)), and nine Mniaceae samples (Figures 1(c), 1(d), and 1(e)). The characteristic peaks of the FTIR spectra obtained from the samples are shown at the bottom. Most of these peaks represent major functional groups, which show the comprehensive information of protein, carbohydrate, fibrin, lipid, and so forth. The FTIR spectra comparison can provide information on the different samples. Hence, the region between 4000 cm−1 and 3000 cm−1 presents a broad band centered at approximately 3380 cm−1 corresponding to the transmittance due to the stretching of O–H bands (Figure 1) [14]. A sharp peak at 2920 cm−1 is due to the presence of polysaccharide, lipid, and carbohydrates (C–H stretch) [15]. The peak at 1640 cm−1 is attributed to the transmittance of amide (N–H bend). A second amide vibration is shown at 1380 cm−1 (C–N stretch), followed by amide peak at 1250 cm−1 (C–N stretch). The peak at 1040 cm−1 can be attributed to oligosaccharides, glycoprotein, and cellulose (C–O stretch) [17]. The peaks from 1200 cm−1 to 400 cm−1 can be attributed to the transmittance of low-molecular weight carbohydrates, polyols, and monosaccharides; this region is characterized as the fingerprint region [15].FTIR spectra obtained in the range of 4000 cm−1 to 400 cm−1 for the four species of family Mielichhoferiaceae (a), three species of family Bryaceae (b), and nine species of family Mniaceae (c), (d), (e); the characteristic peaks are indicated at the bottom.
(a)
(b)
(c)
(d)
(e)


### 4.3. Cluster Analysis

The FTIR spectra from the different species of mosses exhibited similar transmittance. Specific differences were difficult to distinguish by experience. Therefore, we chose multivariate statistical methods to analyze the transmittance bands. Cluster analysis was conducted to investigate the relationships between these taxa. The spectra were classified with the use of the nearest neighbor and Pearson correlation according to the transmittance peaks from 4000 cm−1 to 400 cm−1.The dendrogram divides the 16 samples into two separated clusters (Figure2): cluster 1 (C1) comprises the Mielichhoferiaceae and Bryaceae species and cluster 2 (C2) comprises the Minaceae species. C1 is split into two second subclusters. Subcluster 1 (SC1) comprisesBryum argenteum,B. capillare,andB. uliginosum. Subcluster 2 (SC2) contains the three species of genusPohliain Mielichhoferiaceae. SC1 and SC2 are close to each other. C2 comprises two subclusters. Sc1 comprises the threeMnium species and fourPlagiomnium species; Sc2 contains the twoTrachycystis species (Figure 2). In SC2,Pohlia cruda is clustered withP. longicollis. The result is inconsistent with the molecular result thatP. cruda is distantly related to the other species of genusPohlia.Figure 2
Rescaled distance cluster combine.The result basically reflects the relative relationships of the 16 samples; further study is needed for the internal relationships of the genera. Thus, PCA, FSD, and one-dimensional DWT were adopted in our study.


### 4.4. PCA

We used PCA as the second multivariate analysis. The data of the transmittance peaks in the range of 4000 cm−1 to 400 cm−1 in FTIR spectra were analyzed by PCA. Table 4 shows the variance accounted by the first four PCs computed from the transmittance value of characteristic peaks used in cluster analysis. The first three PCs summarize more variation in the data than any other PCs, accounting for more than 98.58% of the data variance. Figure 3 shows the 3D scores plot based on the first three PCs. It indicates directly that the species of family Bryaceae, Mielichhoferiaceae (A, Figure 3), and Mniaceae (B, Figure 3) can be grouped in two separate ellipses. The studied species of family Mielichhoferiaceae and Bryaceae form two groups (Figure 3). TheTrachycystis species (15, 16) exhibit a short-distance relationship with genusPlagioniumandMnium (Figure 3). The molecular evidence indicated that the genusPohlia was more closely related to family Mniaceae. By contrast, the cluster analysis and PCA data suggest that the species of genusPohlia (1–4) are more closely related to the species of family Bryaceae (5–7). According to the scatter plots, the species of family Bryaceae is nearer to Mielichhoferiaceae than Mniaceae. Combined with cluster analysis, the PCA results can be used to give rough results of classification among the genera simply.Table 4
Variance and cumulative variance for the first four PCs from PCA on the 16 moss species data subsets.
PC
Variance (%)
Cumulative variance (%)
PC1
83.332
83.332
PC2
10.784
94.116
PC3
4.461
98.577
PC4
0.767
99.344Figure 3
3D plot of FTIR spectra of the 16 moss species based on PCA.


### 4.5. FSD Analysis of FTIR Spectral Data

FSD can be used to distinguish the small difference of the functional groups in the fingerprint region [15]. FSD will not change the position and area of the peaks. Besides making the signal peaks narrower, the (1800 to 400) cm−1 range includes the fingerprint region that contains more molecule structure information. Therefore, we use the region (1800 to 400) cm−1 for the FSD extraction of spectral features.Pohlia cruda(1),Bryum argenteum(5),andMnium spinosum(8) are the type species of generaPohlia,Bryum andMnium. In order to further identify them, FSD is used to deal with the FTIR spectra of the three species. The results of FTIR-FSD spectra in the fingerprint region between 1800 and 400 cm−1 are displayed in Figure 4, which shows that the output waveforms of the three species have a marked variation. Certain differences are observed among the three species at the same resolution degree. The C=O bend at 1690 cm−1 is found inBryum argenteumandMnium spinosumbut not inPohlia cruda. The shape of transmittance bands at 1610 cm−1 (C=N) and 1312 cm−1 (N=O) is different. The shape of transmittance bands and the intensity of transmittance peaks in the fingerprint region are quite different. Thus, FSD can be used to easily classify species in different genera.Figure 4
FSD-FTIR spectra in the 1800–400 cm−1 region in the whole plants ofPohlia cruda(1),Bryum argenteum(5),andMnium spinosum(8). Characteristic transmittance bands are indicated at the bottom.


### 4.6. Wavelet Analysis of FTIR Spectral Data

Species that belong to the same genus contain similar chemical components (e.g., protein, carbohydrate, and plant hormones) [40]. FTIR spectra of the same genus exhibit close transmittance values at the same wavenumber. For instance, the FTIR transmittances of the two species in genusMnium are quite difficult to distinguish. The 1300 cm−1 to 400 cm−1 region can usually provide higher characteristic information of the spectra [14]. This fingerprint region contains more molecular structural information. Therefore, in our study, DWT was used to extract their features for further classification.One-dimensional DWT was applied to decompose the FTIR spectral data ofMnium laevinerve(9) andM. marginatum(10) into different frequency bands. The vibration signals were then decomposed up to five levels using Daubechies 4 mother wavelet. The DWT coefficients effectively reflect the features of spectra (Figure 5). Scale 1 contains large noise, which is unsuitable in analyzing the difference of the same genus; determining the differences from Scale 5 is difficult as well. Scales 2–4 show an obvious difference between the twoMniumspecies. The DWT coefficients of the whole plant ofMnium laevinerve(9) andM. marginatum(10) are different. Therefore, the decomposition levels 2–4 in the DWT domain are chosen as a variable characteristic extraction region. In Scales 2–4, different species in the same genus can be simply and effectively identified. The results prove that DWT can be used to extract the features of the FTIR transmittances of the plants in the same genus. Thus, DWT can be used to subdivide bryophytes species.Results of the multiresolution decomposition for the twoMnium species. FTIR spectra with DWT. (a)Mnium laevinerve(9) and (b)M. marginatum(10).
(a)
(b)


## 4.1. Validation of the Method

### 4.1.1. Precision Test

The precision test was conducted by replicate measurements of the same sample (Plagiomnium acutum) for five times in a day. The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤0.51%.Table 3
FTIR transmittances of the characteristic common peaks of precision and repeatability tests onPlagiomnium  acutum.
Peak number
Transmittance % mean (RSD %)
PT
RT
1
13.386 (0.51)
13.635 (0.81)
2
28.041 (0.14)
29.163 (0.18)
3
52.856 (0.12)
53.932 (2.17)
4
13.969 (0.23)
14.306 (0.76)
5
22.608 (0.16)
21.652 (0.84)
6
10.738 (0.33)
10.560 (1.42)
7
29.045 (0.13)
30.145 (0.73)
8
27.033 (0.12)
27.145 (0.95)
9
25.063 (0.15)
25.852 (1.07)
10
23.617 (0.09)
24.444 (1.17)
Precision test (PT,n
=
5), repeatability test (RT, n
=
5).


### 4.1.2. Repeatability Test

The repeatability of the method was assessed by analyzing five independently prepared samples ofPlagiomnium acutumfrom five different places using the same method (Table 2). The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤2.17%.The running results show that the FTIR transmittances of the characteristic common peaks of the same species are stable. The method is reliable and exhibits good repeatability; thus, it can be applied in the analysis of other moss samples.


## 4.1.1. Precision Test

The precision test was conducted by replicate measurements of the same sample (Plagiomnium acutum) for five times in a day. The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤0.51%.Table 3
FTIR transmittances of the characteristic common peaks of precision and repeatability tests onPlagiomnium  acutum.
Peak number
Transmittance % mean (RSD %)
PT
RT
1
13.386 (0.51)
13.635 (0.81)
2
28.041 (0.14)
29.163 (0.18)
3
52.856 (0.12)
53.932 (2.17)
4
13.969 (0.23)
14.306 (0.76)
5
22.608 (0.16)
21.652 (0.84)
6
10.738 (0.33)
10.560 (1.42)
7
29.045 (0.13)
30.145 (0.73)
8
27.033 (0.12)
27.145 (0.95)
9
25.063 (0.15)
25.852 (1.07)
10
23.617 (0.09)
24.444 (1.17)
Precision test (PT,n
=
5), repeatability test (RT, n
=
5).


## 4.1.2. Repeatability Test

The repeatability of the method was assessed by analyzing five independently prepared samples ofPlagiomnium acutumfrom five different places using the same method (Table 2). The results are shown in Table 3. The RSD of the FTIR transmittances of the communal peaks is ≤2.17%.The running results show that the FTIR transmittances of the characteristic common peaks of the same species are stable. The method is reliable and exhibits good repeatability; thus, it can be applied in the analysis of other moss samples.


## 4.2. FTIR Analysis

The FTIR spectra of the 16 samples of mosses were recorded in three sets: four Mielichhoferiaceae samples (Figure1(a)), three Bryaceae samples (Figure 1(b)), and nine Mniaceae samples (Figures 1(c), 1(d), and 1(e)). The characteristic peaks of the FTIR spectra obtained from the samples are shown at the bottom. Most of these peaks represent major functional groups, which show the comprehensive information of protein, carbohydrate, fibrin, lipid, and so forth. The FTIR spectra comparison can provide information on the different samples. Hence, the region between 4000 cm−1 and 3000 cm−1 presents a broad band centered at approximately 3380 cm−1 corresponding to the transmittance due to the stretching of O–H bands (Figure 1) [14]. A sharp peak at 2920 cm−1 is due to the presence of polysaccharide, lipid, and carbohydrates (C–H stretch) [15]. The peak at 1640 cm−1 is attributed to the transmittance of amide (N–H bend). A second amide vibration is shown at 1380 cm−1 (C–N stretch), followed by amide peak at 1250 cm−1 (C–N stretch). The peak at 1040 cm−1 can be attributed to oligosaccharides, glycoprotein, and cellulose (C–O stretch) [17]. The peaks from 1200 cm−1 to 400 cm−1 can be attributed to the transmittance of low-molecular weight carbohydrates, polyols, and monosaccharides; this region is characterized as the fingerprint region [15].FTIR spectra obtained in the range of 4000 cm−1 to 400 cm−1 for the four species of family Mielichhoferiaceae (a), three species of family Bryaceae (b), and nine species of family Mniaceae (c), (d), (e); the characteristic peaks are indicated at the bottom.
(a)
(b)
(c)
(d)
(e)


## 4.3. Cluster Analysis

The FTIR spectra from the different species of mosses exhibited similar transmittance. Specific differences were difficult to distinguish by experience. Therefore, we chose multivariate statistical methods to analyze the transmittance bands. Cluster analysis was conducted to investigate the relationships between these taxa. The spectra were classified with the use of the nearest neighbor and Pearson correlation according to the transmittance peaks from 4000 cm−1 to 400 cm−1.The dendrogram divides the 16 samples into two separated clusters (Figure2): cluster 1 (C1) comprises the Mielichhoferiaceae and Bryaceae species and cluster 2 (C2) comprises the Minaceae species. C1 is split into two second subclusters. Subcluster 1 (SC1) comprisesBryum argenteum,B. capillare,andB. uliginosum. Subcluster 2 (SC2) contains the three species of genusPohliain Mielichhoferiaceae. SC1 and SC2 are close to each other. C2 comprises two subclusters. Sc1 comprises the threeMnium species and fourPlagiomnium species; Sc2 contains the twoTrachycystis species (Figure 2). In SC2,Pohlia cruda is clustered withP. longicollis. The result is inconsistent with the molecular result thatP. cruda is distantly related to the other species of genusPohlia.Figure 2
Rescaled distance cluster combine.The result basically reflects the relative relationships of the 16 samples; further study is needed for the internal relationships of the genera. Thus, PCA, FSD, and one-dimensional DWT were adopted in our study.


## 4.4. PCA

We used PCA as the second multivariate analysis. The data of the transmittance peaks in the range of 4000 cm−1 to 400 cm−1 in FTIR spectra were analyzed by PCA. Table 4 shows the variance accounted by the first four PCs computed from the transmittance value of characteristic peaks used in cluster analysis. The first three PCs summarize more variation in the data than any other PCs, accounting for more than 98.58% of the data variance. Figure 3 shows the 3D scores plot based on the first three PCs. It indicates directly that the species of family Bryaceae, Mielichhoferiaceae (A, Figure 3), and Mniaceae (B, Figure 3) can be grouped in two separate ellipses. The studied species of family Mielichhoferiaceae and Bryaceae form two groups (Figure 3). TheTrachycystis species (15, 16) exhibit a short-distance relationship with genusPlagioniumandMnium (Figure 3). The molecular evidence indicated that the genusPohlia was more closely related to family Mniaceae. By contrast, the cluster analysis and PCA data suggest that the species of genusPohlia (1–4) are more closely related to the species of family Bryaceae (5–7). According to the scatter plots, the species of family Bryaceae is nearer to Mielichhoferiaceae than Mniaceae. Combined with cluster analysis, the PCA results can be used to give rough results of classification among the genera simply.Table 4
Variance and cumulative variance for the first four PCs from PCA on the 16 moss species data subsets.
PC
Variance (%)
Cumulative variance (%)
PC1
83.332
83.332
PC2
10.784
94.116
PC3
4.461
98.577
PC4
0.767
99.344Figure 3
3D plot of FTIR spectra of the 16 moss species based on PCA.


## 4.5. FSD Analysis of FTIR Spectral Data

FSD can be used to distinguish the small difference of the functional groups in the fingerprint region [15]. FSD will not change the position and area of the peaks. Besides making the signal peaks narrower, the (1800 to 400) cm−1 range includes the fingerprint region that contains more molecule structure information. Therefore, we use the region (1800 to 400) cm−1 for the FSD extraction of spectral features.Pohlia cruda(1),Bryum argenteum(5),andMnium spinosum(8) are the type species of generaPohlia,Bryum andMnium. In order to further identify them, FSD is used to deal with the FTIR spectra of the three species. The results of FTIR-FSD spectra in the fingerprint region between 1800 and 400 cm−1 are displayed in Figure 4, which shows that the output waveforms of the three species have a marked variation. Certain differences are observed among the three species at the same resolution degree. The C=O bend at 1690 cm−1 is found inBryum argenteumandMnium spinosumbut not inPohlia cruda. The shape of transmittance bands at 1610 cm−1 (C=N) and 1312 cm−1 (N=O) is different. The shape of transmittance bands and the intensity of transmittance peaks in the fingerprint region are quite different. Thus, FSD can be used to easily classify species in different genera.Figure 4
FSD-FTIR spectra in the 1800–400 cm−1 region in the whole plants ofPohlia cruda(1),Bryum argenteum(5),andMnium spinosum(8). Characteristic transmittance bands are indicated at the bottom.


## 4.6. Wavelet Analysis of FTIR Spectral Data

Species that belong to the same genus contain similar chemical components (e.g., protein, carbohydrate, and plant hormones) [40]. FTIR spectra of the same genus exhibit close transmittance values at the same wavenumber. For instance, the FTIR transmittances of the two species in genusMnium are quite difficult to distinguish. The 1300 cm−1 to 400 cm−1 region can usually provide higher characteristic information of the spectra [14]. This fingerprint region contains more molecular structural information. Therefore, in our study, DWT was used to extract their features for further classification.One-dimensional DWT was applied to decompose the FTIR spectral data ofMnium laevinerve(9) andM. marginatum(10) into different frequency bands. The vibration signals were then decomposed up to five levels using Daubechies 4 mother wavelet. The DWT coefficients effectively reflect the features of spectra (Figure 5). Scale 1 contains large noise, which is unsuitable in analyzing the difference of the same genus; determining the differences from Scale 5 is difficult as well. Scales 2–4 show an obvious difference between the twoMniumspecies. The DWT coefficients of the whole plant ofMnium laevinerve(9) andM. marginatum(10) are different. Therefore, the decomposition levels 2–4 in the DWT domain are chosen as a variable characteristic extraction region. In Scales 2–4, different species in the same genus can be simply and effectively identified. The results prove that DWT can be used to extract the features of the FTIR transmittances of the plants in the same genus. Thus, DWT can be used to subdivide bryophytes species.Results of the multiresolution decomposition for the twoMnium species. FTIR spectra with DWT. (a)Mnium laevinerve(9) and (b)M. marginatum(10).
(a)
(b)


## 5. Conclusion

In recent years, the phylogenetic relationships of family Mielichhoferiaceae, Bryaceae, and Mniaceae are still unclear. The traditional circumscriptions are based mainly on peristome characters, making the classification of the three taxa difficult. Results of the present analysis show that FTIR spectroscopy in combination with PCA and cluster analyses can be used to discriminate the genera in Mielichhoferiaceae, Bryaceae, and Mniaceae. Recent studies based on molecular data indicated that genusPohlia belongs to the family Mniaceae [41]. In our study, we find that genusPohlia is more related to Bryaceae with a good support using chemical methods on the basis of their different chemical composition. The PCA results show that thePohliaspecies are not closely related to the species of Mniaceae, and the three families can be basically separated from each other. FSD and DWT are used to extract the features and enhance the differences between the species in the fingerprint region. The twoMniumspecies are successfully identified by FTIR spectroscopy method combined with DWT. Results of the FTIR spectroscopy combined with cluster analyses and PCA can also be used to identify different genera and families of mosses; FTIR spectroscopy method combined with DWT is suitable for discriminating different species of the same genus. The results show the possibility of the use of optical methods, such as FTIR method, in differentiating the genera and species of mosses. FTIR combined with chemometric methods for identification of mosses is rapid and efficient, and this could enable routine laboratories to facilitate identification procedures on mosses with the same technology.



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*Source: 191796-2014-07-14.xml*